Haemophilus adhesion proteins

ABSTRACT

The invention relates to novel Haemophilus adhesion proteins, nucleic acids, and antibodies.

This application was filed under 35 U.S.C. § 371 from PCT/US96/04031, filed Mar. 22, 1996, which was a continuation in part of U.S. application Ser. No. 08/409,995, filed Mar. 24, 1995, now U.S. Pat. No. 5,646,259.

The U.S. Government has certain rights in this invention pursuant to grant numbers AI-21707 and HD-29687 from National Institutes of Health.

FIELD OF THE INVENTION

The invention relates to novel Haemophilus adhesion proteins, nucleic acids, and antibodies.

BACKGROUND OF THE INVENTION

Most bacterial diseases begin with colonization of a particular mucosal surface (Beachey et al., 1981, J. Infect. Dis. 143:325-345). Successful colonization requires that an organism overcome mechanical cleansing of the mucosal surface and evade the local immune response. The process of colonization is dependent upon specialized microbial factors that promote binding to host cells (Hultgren et al., 1993 Cell, 73:887-901). In some cases the colonizing organism will subsequently enter (invade) these cells and survive intracellularly (Falkow, 1991, Cell 65:1099-1102).

Haemophilus influenzae is a common commensal organism of the human respiratory tract (Kuklinska and Kilian, 1984, Eur. J. Clin. Microbiol. 3:249-252). It is the most common cause of bacterial meningitis and a leading cause of other invasive (bacteraemic) diseases. In addition, this organism is responsible for a sizeable fraction of acute and chronic otitis media, sinusitis, bronchitis, and pneumonia.

Haemophilus influenzae is a human-specific organism that normally resides in the human nasopharynx and must colonize this site in order to avoid extinction. This microbe has a number of surface structures capable of promoting attachment to host cells (Guerina et al., 1982, J. Infect. Dis. 146:564; Pichichero et al., 1982, Lancet ii:960-962; St. Geme et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:2875-2879). In addition, H. influenzae has acquired the capacity to enter and survive within these cells (Forsgren et al., 1994, Infect. Immun. 62:673-679; St. Geme and Falkow, 1990, Infect. Immun. 58:4036-4044; St. Geme and Falkow, 1991, Infect. Immun. 59:1325-1333, Infect. Immun. 59:3366-3371). As a result, this bacterium is an important cause of both localized respiratory tract and systemic disease (Turk, 1984, J. Med. Microbiol. 18:1-16). Nonencapsulated, non-typable strains account for the majority of local disease (Turk, 1984, supra); in contrast, serotype b strains, which express a capsule composed of a polymer of ribose and ribitol-5-phosphate (PRP), are responsible for over 95% of cases of H. influenzae systemic disease (Turk, 1982. Clinical importance of Haemophilus influenzae, p. 3-9. In S. H. Sell and P. F. Wright (ed.), Haemophilus influenzae epidemiology, immunology, and prevention of disease. Elsevier/North-Holland Publishing Co., New York).

The initial step in the pathogenesis of disease due to H. influenzae involves colonization of the upper respiratory mucosa (Murphy et al., 1987, J. Infect. Dis. 5:723-731). Colonization with a particular strain may persist for weeks to months, and most individuals remain asymptomatic throughout this period (Spinola et al., 1986, I. Infect. Dis. 154:100-109). However, in certain circumstances colonization will be followed by contiguous spread within the respiratory tract, resulting in local disease in the middle ear, the sinuses, the conjunctiva, or the lungs. Alternatively, on occasion bacteria will penetrate the nasopharyngeal epithelial barrier and enter the bloodstream.

In vitro observations and animal studies suggest that bacterial surface appendages called pili (or fimbriae) play an important role in H. influenzae colonization. In 1982 two groups reported a correlation between piliation and increased attachment to human oropharyngeal epithelial cells and erythrocytes (Guerina et al., supra; Pichichero et al., supra). Other investigators have demonstrated that anti-pilus antibodies block in vitro attachment by piliated H. influenzae (Forney et al., 1992, J. Infect. Dis. 165:464-470;van Alphen et al., 1988, Infect. Immun.56:1800-1806). Recently Weber et al. insertionally inactivated the pilus structural gene in an H. influenzae type b strain and thereby eliminated expression of pili; the resulting mutant exhibited a reduced capacity for colonization of year-old monkeys (Weber et al., 1991, Infect. Immun. 59:4724-4728).

A number of reports suggest that nonpilus factors also facilitate Haemophilus colonization. Using the human nasopharyngeal organ culture model, Farley et al. (1986, J. Infect. Dis. 161:274-280) and Loeb et al. (1988, Infect. Immun. 49:484-489) noted that nonpiliated type b strains were capable of mucosal attachment. Read and coworkers made similar observations upon examining nontypable strains in a model that employs nasal turbinate tissue in organ culture (1991, J. Infect. Dis. 163:549-558). In the monkey colonization study by Weber et al. (1991, supra), nonpiliated organisms retained a capacity for colonization, though at reduced densities; moreover, among monkeys originally infected with the piliated strain, virtually all organisms recovered from the nasopharynx were nonpiliated. All of these observations are consistent with the finding that nasopharyngeal isolates from children colonized with H. influenzae are frequently nonpiliated (Mason et al., 1985, Infect. Immun. 49:98-103; Brinton et al., 1989, Pediatr. Infect. Dis. J. 8:554-561) Previous studies have shown that H. influenzae are capable of entering (invading) cultured human epithelial cells via a pili-independent mechanism (St. Geme and Falkow, 1990, supra; St. Geme and Falkow, 1991. supra). Although H. influenzae is not generally considered an intracellular parasite, a recent report suggests that these in vitro findings may have an in vivo correlate (Forsgren et al., 1994, supra). Forsgren and coworkers examined adenoids from 10 children who had their adenoids removed because of longstanding secretory otitis media or adenoidal hypertrophy. In all 10 cases there were viable intracellular H. influenzae. Electron microscopy demonstrated that these organisms were concentrated in the reticular crypt epithelium and in macrophage-like cells in the subepithelial layer of tissue. One possibility is that bacterial entry into host cells provides a mechanism for evasion of the local immune response, thereby allowing persistence in the respiratory tract.

Thus, a vaccine for the therapeutic and prophylactic treatment of Haemophilus infection is desirable. Accordingly, it is an object of the present invention to provide for recombinant Haemophilus Adherence (HA) proteins and variants thereof, and to produce useful quantities of these HA proteins using recombinant DNA techniques.

It is a further object of the invention to provide recombinant nucleic acids encoding HA proteins, and expression vectors and host cells containing the nucleic acid encoding the HA protein.

An additional object of the invention is to provide monoclonal antibodies for the diagnosis of Haemophilus infection.

A further object of the invention is to provide methods for producing the HA proteins, and a vaccine comprising the HA proteins of the present invention. Methods for the therapeutic and prophylactic treatment of Haemophilus infection are also provided.

SUMMARY OF THE INVENTION

In accordance with the foregoing objects, the present invention provides recombinant HA proteins, and isolated or recombinant nucleic acids which encode the HA proteins of the present invention. Also provided are expression vectors which comprise DNA encoding a HA protein operably linked to transcriptional and translational regulatory DNA, and host cells which contain the expression vectors.

The invention provides also provides methods for producing HA proteins which comprises culturing a host cell transformed with an expression vector and causing expression of the nucleic acid encoding the HA protein to produce a recombinant HA protein.

The invention also includes vaccines for Haemophilus influenzae infection comprising an HA protein for prophylactic or therapeutic use in generating an immune response in a patient. Methods of treating or preventing Haemophilus influenzae infection comprise administering a vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C (SEQ ID NO:1) depict the nucleic acid sequence of HA1.

FIGS. 2A-2D (SEQ ID NO:2) depict the amino acid sequence of HA1.

FIGS. 3A, 3B, 3C, 3D, 3E, 3F and 3G (SEQ ID NOS: 3 and 4) depict the nucleic acid sequence and amino acid sequence of HA2.

FIG. 4 shows the schematic alignment of HA1 and HA2. Regions of sequence similarity are indicated by shaded, striped, and open bars, corresponding to N-terminal domains, internal domains, and C-terminal domains, respectively. The solid circles represent a conserved Walker box ATP-binding motif (GINVSGKT). Numbers above the bars refer to amino acid residue positions in the full-length proteins. Numbers in parentheses below the HA2 bars represent percent similarity/percent identity between these domains and the corresponding HA1 domains. The regions of HA2 defined by amino acid residues 51 to 173, 609 to 846, and 1292 to 1475 show minimal similarity to amino acids 51 to 220 of HA1.

FIGS. 5A and 5B (SEQ ID NOS: 5 and 6) depict the homology between the N-terminal amino acid sequences of HA1 and HA2. Single letter abbreviations are used for the amino acids. A line indicates identity between the residues, and two dots indicate conservative changes, i.e. similarity between residues.

FIG. 6 depicts the restriction maps of phage 11-17 and plasmid pT7-7 subclones.

FIG. 7 depicts the restriction map of pDC400 and derivatives. pDC400 contains a 9.1 kb insert from strain C54 cloned into pUC19. Vector sequences are represented by hatched boxes. Letters above the top horizontal line indicate restriction enzyme sites: Bg, BglII; E, EcoRi; H, HindlIl; P, Pst; S, Sail; Ss, SstI; X,XbaI. The heavy horizontal line with arrow represents the location of the hsf locus within pDC400 and the direction of transcription. The striated horizontal line represents the 3.3 kb intragenic fragment used as a probe for Southern analysis. The plasmid pDC602, which is not shown, contains the same insert as pDC601, but in the opposite orientation.

FIG. 8 shows the identification of plasmid-encoded proteins using the bacteriophage T7 expression system. Bacteria were radiolabelled with trans-[³⁵S]-label, and whole cell lysates were resolved on a 7.5% SDS-polyacrylamidegel. Proteins were visualized by autoradiography. Lane 1, E. coli BL21(DE3)/pT7-7uninduced; lane 2, BL21(DE3)/pT7-7 induced; lane 3, BL21(DE3)/pDC602 uninduced; lane 4, BL21(DE3)/pDC602 induced; lane 5, BL21(DE3)/pDC601 uninduced; lane 6, BL21(DE3)/pDC601 induced. The plasmids pDC602 and pDC601 are derivatives of pT7-7 that contain the 8.3 kbXbal fragment from pDC400 in opposite orientations. The asterisk indicates the overexpressed protein in BL21 (DE3)/pDC601.

FIGS. 9A and 9B depict the southern analysis of chromosomal DNA from H. influenzae strains C54 and 11, probing with HA2 versus HA 1. DNA fragments were separated on a 0.7% agarose gel and transferred bidirectionally to nitrocellulose membranes prior to probing with either HA1 or HA2. Lane 1, C54 chromosomal DNA digested with BglII; lane 2, C54 chromosomal DNA digested with ClaI; lane 3, C54 chromosomal DNA digested with PstI; lane 4, 11 chromosomal DNA digested with BglII; lane 5, 11 chromosomal DNA digested with ClaI; lane 6, 11 chromosomal DNA digested with XbaI. A. Hybridization with the 3.3 kb PstI-BglII intragenic fragment of HA2 from strain C54. B. Hybridization with the 1.6 kb StyI-SspI intragenic fragment of HA1 from strain 11.

FIG. 10 depicts the comparison of cellular binding specificities of E. coli DH5α harboring HA2 versus HA1. Adherence was measured after incubating bacteria with eucaryotic cell monolayers for 30 minutes as described and was calculated by dividing the number of adherent colony forming units by the number of inoculated colony forming units (St. Geme et al., 1993). Values are the mean ±SEM of measurements made in triplicate from representative experiments. The plasmid pDC601 contains the HA2 gene from H. influenzae strain C54, while pHMW8-5 contains the HA I gene from nontypable H. influenzae strain 11. Both pDC601 and pHMW8-5 were prepared using pT7-7 as the cloning vector.

FIG. 11 depicts the comparison of the N-terminal extremities of HA2 (SEQ ID NO:7), HMW 1 (SEQ ID NO:9), HMW2 (SEQ ID NO:10), AIDA-I (SEQ ID NO:11), Tsh (SEQ ID NO:12), and SepA (SEQ ID NO:13). The N-terminal sequence of HA2 (SEQ ID NO:7) is aligned with those of HA1 (Barenkamp, S. J., and J. W. St. Geme, III. Identification of a second family of high molecular weight adhesion proteins expressed by nontypable Haemophilus influenzae. Mol. Microbiol., in press.), HMW1 (SEQ ID NO:9) and HMW2 (SEQ ID NO:10) (Barenkamp, S. J., and E. Leininger. 1992. Cloning, expression, and DNA sequence analysis of genes encoding nontypeable Haemophilus influenzae high molecular weight surface-exposed proteins related to filamentoushemagglutinin of Bordetella pertussis. Infect. Immun. 60:1302-1313.), AIDA-I (SEQ ID NO:11) (Benz, I., and M. A. Schmidt. 1992. AIDA-I (SEQ ID NO:11), the adhesin involved in diffuse adherence of the diarrhoeagenic Escherichia coli strain 2787 (0126:H27), is synthesized via a precursor molecule. Mol. Microbiol. 6:1539-1546.), Tsh (SEQ ID NO:12) (Provence, D. and R. Curtiss III. 1994. Isolation and characterization of a gene involved in hemagglutination by an avian pathogenic Escherichia coli strain. Infect. Immun. 62:1369-1380.), and SepA (SEQ ID NO:13) (Benjelloun-Touimi, Z., P. J. Sansonetti, and C. Parsot. 1995. SepA (SEQ ID NO:13), the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion. Mol. Microbiol. 17:123-135.). A consensus sequence is shown on the lower line.

FIG. 12 depicts the southern analysis of chromosomal DNA from epidemiologically distinct strains of H. influenzae type b. Chromosomal DNA was digested with BglII, separated on a 0.7% agarose gel, transferred to nitrocellulose, and probed with the 3.3 kb PstI-BglII intragenic fragment of hsf from strain C54. Lane 1, strain C54; lane 2, strain 1081; lane 3, strain 1065; lane 4, strain 1058; lane 5, strain 1060; lane 6, strain 1053; lane 7, strain 1063; lane 8, strain 1069; lane 9, strain 1070; lane 10, strain 1076; lane 11, strain 1084.

FIG. 13 depicts the southern analysis of chromosomal DNA from non-type b encapsulated strains of H. influenzae. Chromosomal DNA was digested with BglII, separated on a 0.7% agarose gel, transferred to nitrocellulose, and probed with the 3.3 kb PstI-BglII intragenic fragment of hsf from strain C54. Lane 1, SM4 (type a); lane 2, SM72 (type c); lane 3, SM6 (type d); lane 4, Rd (type d); lane 5, SM7 (type e); lane 6, 142 (type e); lane 7, 327 (type e); lane 8, 351 (type e); lane 9, 134 (type f); lane 10, 219 (type f). lane 11, 346 (type f; lane 12, 503 (type f).

FIGS. 14A and 14B (SEQ ID NO:14) are the nucleic acid sequence of HA3.

FIG. 15 (SEQ ID NO:15) is the amino acid sequence of HA3.

FIGS. 16A and 16B (SEQ ID NOS: 2 and 15) depict the homology between the amino acid sequences of HA1 and HA3. Single letter abbreviations are used for the amino acids. A line indicates identity between the residues, and two dots indicate conservative changes, i.e. similarity between residues.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel Haemophilus Adhesion (HA) proteins. In a preferred embodiment, the HA proteins are from Haemophilus strains, and in the preferred embodiment, from Haemophilus influenza. In particular. H. influenzae encapsulated type b strains are used to clone the HA proteins of the invention. However, using the techniques outlined below, HA proteins from other Haemophilus influenzae strains, or from other bacterial species such as Neisseria spp. or Bordetalla spp. may also be obtained.

Three HA proteins, HA1, HA2 and HA3, are depicted in FIGS. 2 (SEQ ID NO:2), 3 (SEQ ID NOS: 3 and 4) and 15(SEQ ID NO:15), respectively. HA2 is associated with the formation of surface fibrils, which are involved in adhesion to various host cells. HA1 has also been implicated in adhesion to a similar set of host cells. When the HA1 or HA2 nucleic acid is expressed in a non-adherent strain of E. coli as described below, the E. coli acquire the ability to adhere to human host cells. It should be noted that in the literature, HA1 is referred to as hia (H. influenza adherence) and HA2 is referred to as hsf (Haemophilus surface fibrils).

A HA protein may be identified in several ways. A HA nucleic acid or HA protein is initially identified by substantial nucleic acid and/or amino acid sequence homology to the sequences shown in FIGS. 1 (SEQ ID NO:1), 2 (SEQ ID NO:2), 3 (SEQ ID NOS: 3 and 4), 14 (SEQ ID NO:14) or 15(SEQ ID NO:15). Such homology can be based upon the overall nucleic acid or amino acid sequence or portions thereof.

As used herein, a protein is a “HA protein” if the overall homology of the protein sequence to the amino acid sequence shown in FIGS. 2 (SEQ ID NO:2) and/or FIG. 3 (SEQ ID NO:4)and/or FIG. 15 (SEQ ID NO:15) is preferably greater than about 45 to 50%, more preferably greater than about 65% and most preferably greater than 80%. In some embodiments the homology will be as high as about 90 to 95 or 98%. That is, a protein that has at least 50% homology (or greater) to one, two or all three of the amino acid sequences of HA1, HA2 and HA3 is considered a HA protein. This homology will be determined using standard techniques known in the art, such as the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12:387-395 (1984) or the BLASTX program (Altschul et al., J. Mol. Biol. 215:403-410 (1990)). The alignment may include the introduction of gaps in the sequences to be aligned. As noted below, in the comparison of proteins of different lengths, such as HA1 and HA3 with HA2, the homology is determined on the basis of the length of the shorter sequence.

In a preferred embodiment, a HA protein is defined as having significant homology to either the N-terminal region or the C-terminal region, or both, of the HA1. HA2 and HA3 proteins depicted in FIGS. 4, 5 and 15. The N-terminal region of about 50 amino acids is virtually identical as between HA1 and HA3 (98% homology), and as between either HA1 or HA3 and HA2 is 74%. As shown in FIG. 11 (SEQ ID NOS: 7-13), the first 24 amino acids of the N-terminus of HA1 and HA2 has limited homology to several other proteins, but this homology is 50% or less. Thus, a HA protein may be defined as having homology to the N-terminal region of at least about 60%, preferably at least about 70%, and most preferably at least about 80%, with homology as high as 90 or 95% especially preferred. Similarly, the C-terminal region of at least about 75, preferably 100 and most preferably 125 amino acid residues is also highly homologous and can be used to identify a HA protein. As shown in FIG. 16, the homology between the C-terminal 120 or so amino acids of HA1 and HA3 is about 98%. and as between either HA1 or HA3 and HA2 is also about 98%. Thus homology at the C-terminus is a particularly useful way of identifying a HA protein. Accordingly, a HA protein can be defined as having homology to the C-terminal region of at least about 60%, preferably at least about 70%, and most preferably at least about 80%, with homology as high as 90 or 95% especially preferred. In a preferred embodiment, the HA protein has homology to both the N- and C-terminal regions.

In addition, a HA protein may be identified as containing at least one stretch of amino acid homology found at least in the HA1 and HA2 proteins as depicted in FIG. 4. HA2 contains three separate stretchs of amino acids (174 to 608, 847 to 1291, and 1476 to 1914, respectively) that shows significant homology to the region of HA1 defined by amino acids 221 to 658.

The HA proteins of the present invention have limited homology to the high molecular weight protein-1 (HMW1 ) of H. influenzae, as well as the AIDA-I adhesin of E. coli. For the HMW1 protein, this homology is greatest between residues 60-540 of the HA1 protein and residues 1100 to about 1550 of HMW1, with 20% homology in this overlap region. For the AIDA-I protein, there is a roughly 50% homology between the first 30 amino acids of AIDA-I and HA1, and the overall homology between the proteins is roughly 22%.

In addition, the HA1, HA2 and HA3 proteins of the present invention have homology to each other, as shown in FIGS. 4, 5 and 16. As between HA1 and HA2, the homology is 81% similarity and 72% identity overall. HA3 and HA1 are 51% identical and 65% similar. Thus, for the purposes of the invention, HA1, HA2 and HA3 are all HA proteins.

An “HA1” protein is defined by substantial homology to the sequence shown in FIG. 2 (SEQ ID NO:2). This homology is preferably greater than about 60%, more preferably greater than about 70% and most preferably greater than 80%. In preferred embodiments the homology will be as high as about 90 to 95 or 98%. Similarly, an “HA2” protein may be defined by the same substantial homology to the sequence shown in FIG. 3 (SEQ ID NO:4), and a “HA3” protein is defined with reference to FIG. 15 (SEQ ID NO:15), as defined above.

In addition, for sequences which contain either more or fewer amino acids than the proteins shown in FIGS. 2 (SEQ ID NO:2), 3 (SEQ ID NO:4) and 15 (SEQ ID NO:15), it is understood that the percentage of homology will be determined based on the number of homologous amino acids in relation to the total number of amino acids. Thus, for example, homology of sequences shorter than that shown in FIGS. 2 (SEQ ID NO:2), 3 (SEQ ID NO:4) and 15 (SEQ ID NO:15), as discussed below, will be determined using the number of amino acids in the shorter sequence.

HA proteins of the present invention may be shorter than the amino acid sequences shown in FIGS. 2 (SEQ ID NO:2), 3 (SEQ ID NO:4) and 15 (SEQ ID NO:15). Thus, in a preferred embodiment, included within the definition of HA proteins are portions or fragments of the sequence shown in FIGS. 2 (SEQ ID NO:2), 3 (SEQ ID NO:4) and 15 (SEQ ID NO:15). Generally, the HA protein fragments may range in size from about 7 amino acids to about 800 amino acids, with from about 15 to about 700 amino acids being preferred, and from about 100 to about 650 amino acids also preferred. Particularly preferred fragments are sequences unique to HA; these sequences have particular use in cloning HA proteins from other organisms, to generate antibodies specific to HA proteins, or for particular use as a vaccine. Unique sequences are easily identified by those skilled in the art after examination of the HA protein sequence and comparison to other proteins; for example, by examination of the sequence alignment shown in FIGS. 5 (SEQ ID NOS: 5 and 6) and 16 (SEQ ID NOS: 2 and 15) Preferred unique sequences include the N-terminal region of the HA1, HA2 and HA3 sequences, comprising roughly 50 amino acids and the C-terminal 120 amino acids, depicted in FIGS. 2 (SEQ ID NO:2), 3 (SEQ ID NO:4) and 15 (SEQ ID NO:15). HA protein fragments which are included within the definition of a HA protein include N- or C-terminal truncations and deletions which still allow the protein to be biologically active; for example, which still allow adherence, as described below. In addition, when the HA protein is to be used to generate antibodies, for example as a vaccine, the HA protein must share at least one epitope or determinant with the sequences shown in FIGS. 2 (SEQ ID NO:2), 3 (SEQ ID NO:4) and 15 (SEQ ID NO:15). In a preferred embodiment, the epitope is unique to the HA protein; that is, antibodies generated to a unique epitope exhibit little or no cross-reactivity with other proteins. However, cross reactivity with other proteins does not preclude such epitopes or antibodies for immunogenic or diagnostic uses. By “epitope” or “determinant” herein is meant a portion of a protein which will generate and/or bind an antibody. Thus, in most instances, antibodies made to a smaller HA protein will be able to bind to the full length protein.

In some embodiments, the fragment of the HA protein used to generate antibodies are small; thus, they may be used as haptens and coupled to protein carriers to generate antibodies, as is known in the art.

In addition, sequences longer than those shown in FIGS. 2 (SEQ ID NO:2), 3 (SEQ ID NO:4) and 15 (SEQ ID NO: 15) are also included within the definition of HA proteins.

Preferably, the antibodies are generated to a portion of the HA protein which is exposed at the outer membrane, i.e. surface exposed. The amino-terminal portions of HA1, HA2 and HA3 are believed to be externally exposed proteins.

The HA proteins may also be identified as associated with bacterial adhesion. Thus, deletions of the HA proteins from the naturally occuring microorganism such as Haemophilus species results in a decrease or absence of binding ability. In some embodiments, the expression of the HA proteins in a non-adherent bacteria such as E. coli results in the ability of the organism to bind to cells.

In the case of the nucleic acid, the overall homology of the nucleic acid sequence is commensurate with amino acid homology but takes into account the degeneracy in the genetic code and codon bias of different organisms. Accordingly, the nucleic acid sequence homology may be either lower or higher than that of the protein sequence. Thus the homology of the nucleic acid sequence as compared to the nucleic acid sequences of FIGS. 1 (SEQ ID NO:1), 3 (SEQ ID NO:3) and 14 (SEQ ID NO:14)is preferably greater than about 40%, more preferably greater than about 60% and most preferably greater than 80%. In some embodiments the homology will be as high as about 90 to 95 or 98%.

As outlined for the protein sequences, a preferred embodiment utilizes HA nucleic acids with substantial homology to the unique N-terminal and C-terminal regions of the HA1, HA2 and HA3 sequences.

In one embodiment, the nucleic acid homology is determined through hybridization studies. Thus, for example, nucleic acids which hybridize under high stringency to all or part of the nucleic acid sequences shown in FIGS. 1 (SEQ ID NO:1), 3 (SEQ ID NO:3) and 14 (SEQ ID NO:14) are considered HA protein genes. High stringency conditions include, but are not limited to, washes with 0.1XSSC at 65° C. for 2 hours.

The HA proteins and nucleic acids of the present invention are preferably recombinant. As used herein, “nucleic acid” may refer to either DNA or RNA, or molecules which contain both deoxy- and ribonucleotides. The nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids. Specifically included within the definition of nucleic acid are anti-sense nucleic acids. An anti-sense nucleic acid will hybridize to the corresponding non-coding strand of the nucleic acid sequences shown in FIGS. 1 (SEQ ID NO:1), 3 (SEQ ID NO:3) and 14 (SEQ ID NO:14), but may contain ribonucleotides as well as deoxyribonucleotides. Generally, anti-sense nucleic acids function to prevent expression of mRNA, such that a HA protein is not made, or made at reduced levels. The nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence. By the term “recombinant nucleic acid” herein is meant nucleic acid, originally formed in vitro by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature. Thus an isolated HA protein gene, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention; i.e. the HA nucleic acid is joined to other than the naturally occurring Haemophiluschromosome in which it is normally found. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly are still considered recombinant for the purposes of the invention.

Similarly, a “recombinantprotein” is a protein made using recombinanttechniques i.e. through the expression of a recombinant nucleic acid as depicted above. A recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated away from some or all of the proteins and compounds with which it is normally associated in its wild type host, or found in the absence of the host cells themselves. Thus, the protein may be partially or substantially purified. The definition includes the production of a HA protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at increased concentration levels. Alternatively, the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions. Furthermore, although not normally considered “recombinant”, proteins or portions of proteins which are synthesized chemically, using the sequence information of FIGS. 2 (SEQ ID NO:2), 3 (SEQ ID NOS: 3 and 4) and 15 (SEQ ID NO:15), are considered recombinant herein as well.

Also included with the definition of HA protein are HA proteins from other organisms, which are cloned and expressed as outlined below.

In the case of anti-sense nucleic acids, an anti-sense nucleic acid is defined as one which will hybridize to all or part of the corresponding non-coding sequence of the sequences shown in FIGS. 1 (SEQ ID NO:1), 3 (SEQ ID NO:3) and 14 (SEQ ID NO:14). Generally, the hybridization conditions used for the determination of anti-sense hybridization will be high stringency conditions, such as 0.1XSSC at 65° C.

Once the HA protein nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the entire HA protein nucleic acid. Once isolated from its natural source, e.g., contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant HA protein nucleic acid can be further used as a probe to identify and isolate other HA protein nucleic acids. It can also be used as a “precursor” nucleic acid to make modified or variant HA protein nucleic acids and proteins.

Using the nucleic acids of the present invention which encode HA protein, a variety of expression vectors are made. The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the HA protein. “Operably linked” in this context means that the transcriptional and translational regulatory DNA is positioned relative to the coding sequence of the HA protein in such a manner that transcription is initiated. Generally, this will mean that the promoter and transcriptional initiation or start sequences are positioned 5′ to the HA protein coding region. The transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the HA protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus will be used to express the HA protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.

In addition, the expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.

In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.

The HA proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a HA protein, under the appropriate conditions to induce or cause expression of the HA protein. The conditions appropriate for HA protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction. In addition, in some embodiments, the timing of the harvest is important. For example, the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.

Appropriate host cells include yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis SF9 cells. C 129 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells, immortalized mammalian myeloid and lymphoid cell lines.

In a preferred embodiment, HA proteins are expressed in bacterial systems. Bacterial expression systems are well known in the art.

A suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of the coding sequence of HA protein into mRNA. A bacterial promoter has a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful: for example, the tac promoter is a hybrid of the trp and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.

In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable. In E. coli, the ribosome binding site is called the Shine-Delgarno (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon.

The expression vector may also include a signal peptide sequence that provides for secretion of the HA protein in bacteria. The signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell, as is well known in the art. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).

The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.

These components are assembled into expression vectors. Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others.

The bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.

In one embodiment, HA proteins are produced in insect cells. Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art. Briefly, baculovirus is a very large DNA virus which produces its coat protein at very high levels. Due to the size of the baculoviral genome, exogenous genes must be placed in the viral genome by recombination. Accordingly, the components of the expression system include: a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the HA protein; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene into the baculovirus genome); and appropriate insect host cells and growth media.

Mammalian expression systems are also known in the art and are used in one embodiment. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3′) transcription of a coding sequence for HA protein into mRNA. A promoter will have a transcription initiating region, which is usually place proximal to the 5′ end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element, typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, and herpes simplex virus promoter.

Typically transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3′ terminus of the mature mRNA is formed by site-specificpost-translational cleavage and polyadenylation. Examples of transcription terminator and polyadenlytion signals include those derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well known in the art, and will vary with the host cell used. Techniques include dextran-mediated transfection, calcium phosphate precipitation polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s)in liposomes, and direct microinjection of the DNA into nuclei.

In a preferred embodiment, HA protein is produced in yeast cells. Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. Preferred promoter sequences for expression in yeast include the inducible GAL 1,10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and the acid phosphatase gene. Yeast selectable markers include ADE2, HIS4, LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; the G418 resistance gene, which confers resistance to G418; and the CUP 1 gene, which allows yeast to grow in the presence of copper ions.

A recombinant HA protein may be expressed intracellularly or secreted. The HA protein may also be made as a fusion protein, using techniques well known in the art. Thus, for example, if the desired epitope is small, the HA protein may be fused to a carrier protein to form an immunogen. Altematively, the HA protein may be made as a fusion protein to increase expression.

Also included within the definition of HA proteins of the present invention are amino acid sequence variants. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the HA protein, using cassette mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant HA protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the HA protein amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.

While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed HA protein variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis. Screening of the mutants is done using assays of HA protein activities; for example, mutated HA genes are placed in HA deletion strains and tested for HA activity, as disclosed herein. The creation of deletion strains, given a gene sequence, is known in the art. For example, nucleic acid encoding the variants may be expressed in an adhesion deficient strain, and the adhesion and infectivity of the variant Haemophilus influenzae evaluated. For example, as outlined below, the variants may be expressed in the E. coli DH5α non-adherent strain, and the transformed E. coli strain evaluated for adherence using Chang conjunctival cells.

Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to 30 residues, although in some cases deletions may be much larger, as for example when one of the domains of the HA protein is deleted.

Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances.

When small alterations in the characteristics of the HA protein are desired, substitutions are generally made in accordance with the following chart:

CHART I Original Residue Exemplary Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Chart I. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain. e.g. glycine.

The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the polypeptide as needed. Alternatively, the variant may be designed such that the biological activity of the HA protein is altered. For example, the Walker box ATP-binding motif may be altered or eliminated.

In a preferred embodiment, the HA protein is purified or isolated after expression HA proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, the HA protein may be purified using a standard anti-HA antibody column.

Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer-Verlag, N.Y. (1982). The degree of purification necessary will vary depending on the use of the HA protein. In some instances no purification will be necessary.

Once expressed and purified if necessary, the HA proteins are useful in a number of applications.

For example, the HA proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify antibodies from samples obtained from animals or patients exposed to the Haemophilus influenzae organism. The purified antibodies may then be used as outlined below.

Additionally, the HA proteins are useful to make antibodies to HA proteins. These antibodies find use in a number of applications. The antibodies are used to diagnose the presence of an Haemophilus influenzae infection in a sample or patient. In a preferred embodiment, the antibodies are used to detect the presence of nontypable Haemophilus influenza (NTHI). although typable H. influenzae infections are also detected using the antibodies.

This diagnosis will be done using techniques well known in the art; for example, samples such as blood or tissue samples may be obtained from a patient and tested for reactivity with the antibodies, for example using standard techniques such as ELISA. In a preferred embodiment, monoclonal antibodies are generated to the HA protein, using techniques well known in the art. As outlined above, the antibodies may be generated to the full length HA protein, or a portion of the HA protein.

Antibodies generated to HA proteins may also be used in passive immunization treatments, as is known in the art.

Antibodies generated to unique sequences of HA proteins may also be used to screen expression libraries from other organisms to find, and subsequently clone, HA nucleic acids from other organisms.

In one embodiment, the antibodies may be directly or indirectly labelled. By “labelled” herein is meant a compound that has at least one element, isotope or chemical compound attached to enable the detection of the compound. In general labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes. The labels may be incorporated into the compound at any position. Thus, for example, the HA protein antibody may be labelled for detection, or a secondary antibody to the HA protein antibody may be created and labelled.

In one embodiment, the antibodies generated to the HA proteins of the present invention are used to purify or separate HA proteins or the Haemophilus influenzae organism from a sample. Thus for example, antibodies generated to HA proteins which will bind to the Haemophilus influenzae organism may be coupled, using standard technology, to affinity chromatography columns. These columns can be used to pull out the Haemophilus organism from environmental or tissue samples.

In a preferred embodiment, the HA proteins of the present invention are used as vaccines for the prophylactic or therapeutic treatment of a Haemophilus influenzae infection in a patient. By “vaccine” or “immnunogenic compositions” herein is meant an antigen or compound which elicits an immune response in an animal or patient The vaccine may be administered prophylactically, for example to a patient never previously exposed to the antigen, such that subsequent infection by the Haemophilus influenzae organism is prevented. Alternatively, the vaccine may be administered therapeutically to a patient previously exposed or infected by the Haemophilus influenzae organism. While infection cannot be prevented, in this case an immune response is generated which allows the patient's immune system to more effectively combat the infection. Thus, for example, there may be a decrease or lessening of the symptoms associated with infection.

A “patient” for the purposes of the present invention includes both humans and other animals and organisms. Thus the methods are applicable to both human therapy and veterinary applications.

The administration of the HA protein as a vaccine is done in a variety of ways. Generally, the HA proteins can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby therapeutically effective amounts of the HA protein are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation are well known in the art. Such compositions will contain an effective amount of the HA protein together with a suitable amount of vehicle in order to prepare pharmaceutically acceptable compositions for effective administration to the host. The composition may include salts, buffers, carrier proteins such as serum albumin, targeting molecules to localize the HA protein at the appropriate site or tissue within the organism, and other molecules. The composition may include adjuvants as well.

In one embodiment, the vaccine is administered as a single dose; that is, one dose is adequate to induce a sufficient immune response to prophylactically or therapeutically treat a Haemophilus influenzae infection. In alternate embodiments, the vaccine is administered as several doses over a period of time, as a primary vaccination and “booster” vaccinations.

By “therapeutically effective amounts” herein is meant an amount of the HA protein which is sufficient to induce an immune response. This amount may be different depending on whether prophylactic or therapeutic treatment is desired. Generally, this ranges from about 0.001 mg to about 1 gm, with a preferred range of about 0.05 to about 0.5 gm. These amounts may be adjusted if adjuvants are used.

The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. All references cited herein are specifically incorporated by reference.

EXAMPLE 1 Cloning of HA1

Many protocols are substantially the same as those outlined in St. Geme et al., Mol. Microbio. 15(1):77-85 (1995).

Bacterial strains, plasmids, and phages

Nontypable H. influenzae strain 11 was the clinical isolate chosen as a prototypic HMW1/HMW2-non-expressingstrain, although a variety of encapsulated typable strains can be used to clone the protein using the sequences of the figures. The organism was isolated in pure culture from the middle ear fluid of a child with acute otitis media. The strain was identified as H. influenzae by standard methods and was classified as nontypable by its failure to agglutinate with a panel of typing antisera for H. influenzae types a to f (Burroughs Wellcome Co., Research Triangle Park, N.C.) and failure to show lines of precipitation with these antisera in counterimmunoelectrophoresis assays. Strain 11 adheres efficiently to Chang conjunctival cells in vitro, at levels comparable to those previously demonstrated for NTHI strains expressing HMW1/HMW2-like proteins (data not shown). Convalescent serum from the child infected with this strain demonstrated an antibody response directed predominantly against surface-exposed high molecular weight proteins with molecular weights greater than 100 kDa.

M13mp 18 and M13mp 19 were obtained from New England BioLabs, Inc. (Beverly, Mass.) pT7-7 was the kind gift of Stanley Tabor. This vector contains the T7 RNA polymerase promoter ø10, a ribosome-binding site, and the translational start site for the T7 gene 10 protein upstream from a multiple cloning site.

Molecular cloning and plasmid subcloning

The recombinant phage containing the HA1 gene was isolated and characterized using methods similar to those described previously. In brief, chromosomal DNA from strain 11 was prepared and Sau3A partial restriction digests of the DNA were prepared and fractionated on 0.7% agarose gels. Fractions containing DNA fragments in the 9- to 20- kbp range were pooled, and a library was prepared by ligation into λEMBL3 arms. Ligation mixtures were packaged in vitro with Gigapack (Stratagene) and plate-amplified in a P2 lysogen of E. coli LE392. Lambda plaque immunological screening was performed as described by Maniatis et al., Molecular Cloning: A Laboratory Manual. 2d Ed. (1989), Cold Spring Harbor Press. For plasmid subcloning studies, DNA from recombinant phage was subcloned into the T7 expression plasmid pT7-7. Standard methods were used for manipulation of cloned DNA as described by Maniatis et al (supra).

Plasmid pHMW8-3 was generated by isolating an 11 kbp Xbal fragment from purified DNA from recombinant phage clone 11-17 and ligating into Xbal cut pT7-7. Plasmid pHMW8-4 was generated by isolating a 10 kbp BamHI-Cial cut pT7-7. Plasmid pHMW8-5 was generated by digesting plasmid pHMW8-3 DNA with Clal, isolating the larger fragment and religating. Plasmid pHMW8-6 was generated by digesting pHMW8-4 with Spel, which cuts at a unique site within the HA1 gene, blunt-ending the resulting fragment, inserting a kanamycin resistance cassette into the Spel site. Plasmidp HMW8-7was generated by digesting pHMW8-3 with Nrul and Hinalll, isolating the fragment containing pT7-7, blunt-ending and religating. The plasmid restriction maps are shown in FIG. 6.

DNA sequence analysis

DNA sequence analysis was performed by the dideoxy method with the U.S. Biochemicals Sequenase kit as suggested by the manufacturer. [³⁶S]dATP was purchased from New England Nuclear (Boston, Mass). Data were analyzed with Compugene software and the Genetics Computer Group program from the University of Wisconsin on a Digital VAX 8530 computer. Several 21-mer oligonucleotide primers were generated as necessary to complete the sequence.

Adherence assays

Adherence assays were done with Chang epithelial cells [Wong-Kilbourne derivative, clone 1-5c4 (human conjunctiva), ATCC CCL20.2)], which were seeded into wells of 24-well tissue culture plates, as described (St. Geme III et al., Infect. Immun. 58:4036(1990)). Bacteria were inoculated into broth and allowed to grow to a density of approximately 2×10⁹ colony-forming units per ml. Approximately 2×10⁷ colony-forming units were inoculated onto epithelial cells monolayers, and plates were gently centrifuged at 165×g for 5 min to facilitate contact between bacteria and the epithelial surface. After incubation for 30 min at 37° C. in 5% CO₂, monolayers were rinsed five times with phosphate buffered saline (PBS) to remove nonadherent organisms and were treated with trypsin-EDTA (0.05% trypsin/0.5% EDTA) in PBS to release them from the plastic support. Well contents were agitated, and dilution were plated on solid medium to yield the number of adherent bacteria per monolayer. Percent adherence was calculated by dividing the number of adherent colony-forming units per monolayer by the number of inoculated colony-forming units.

Isolation and characterization of recombinant phage expressing the strain 11 high molecular weight adhesion protein

The nontypable Haemophilus influenzae strain 11 chromosomal DNA library was screened immunologically with convalescent serum from the child infected with strain 11. Immunoreactive clones were screened by Western blot for expression of high molecular weight proteins with apparent molecular weights>100 dDa and two different classes of recombinant clones were recovered. A single clone designated 11-17 was recovered which expressed the HA1 protein. The recombinant protein expressed by this clone had an apparent molecular weight of greater than 200 kDa.

Transformation into E. coli

Plasmids were introduced into DH5α strain of E. coli (Maniatis, supra), which is a non-adherent strain, using electroporation (Dower et al., Nucl. Acids Res. 16:6127 (1988). The results are shown in Table 1A.

TABLE 1A Strain % Adherence* DH5α(pHMW 8-4) 43.3 ± 5.0% DH5α(pHMW 8-5) 41.3 ± 3.3% DH5α(pHMW 8-6)  0.6 ± 0.3% DH5α(pHMW 8-7) DH5α(pT7-7)  0.4 ± 0.1% *Adherence was measured in a 30 minute assay and was calculated by dividing the number of adherent bacteria by the number of inoculated bacteria. Values are the mean ± SEM of measurements made in triplicate from a representative experiment.

In addition, a monoclonal antibody made by standard procedures, directed against the strain 11 protein recognized proteins in 57 of 60 epidemiologically-unrelated NTHI. However, Southern analysis using the gene indicated that roughly only 25% of the tested strains actually hybridized to the gene (data not shown).

EXAMPLE 2 Cloning of HA2

In a recent study we examined a series of H. influenza type b isolates by transmission electron microscopy and visualized short, thin surface fibrils distinct from pili (St. Geme, J.W. III, and D. Cutter. 1995. Evidence that surface fibrils expressed by Haemophilus influenzae type b promote attachment to human epithelial cells. Mol. Microbiol. 15:77-85.). In that study, the large genetic locus involved in the expression of these appendages was isolated.

Bacterial strains and plasmids

H. influenzae strain C54 is a type b strain that has been described previously (Pichichero. M. E., P. Anderson, M. Loeb, and D. H. Smith. 1982. Do pili play a role in pathogenicity of Haemophilus influenzae type b? Lancet. ii:960-962.). Strain C54-Tn400.23 is a mutant that contains a mini-Tn10 kan element in the hsf locus and demonstrates minimal in vitro adherence (St. Geme, J. W. III, and D. Cutter. 1995. Evidence that surface fibrils expressed by Haemophilus influenzae type b promote attachment to human epithelial cells. Mol. Microbiol.15:77-85.). Strains 1053, 1058, 1060, 1063, 1065, 1069, 1070, 1076, 1081, and 1084 are H. influenzae type b isolates generously provided by J. Musser (Baylor University, Houston, Tex.) (Musser et al., 1990. Global genetic structure and molecular epidemiology of encapsulated Haemophilus influenzae. Rev. Infect. Dis. 12:75-111.). H. influenzae strains SM4 (type a), SM6 (type d), SM7 (type e), and SM72 (type c) are type strains obtained from R. Facklam at the Centers for Disease Control (Atlanta. Ga.). Strains 142,327, and 351 are H. influenzae type e isolates, and strains 134, 219, 256, and 501 are H. influenzae type f isolates obtained from H. Kayhty (Finnish National Public Health Institute, Helsinki). Strain Rd (type d) and the 15 nontypable isolates examined by Southern analysis have been described previously (Alexander et al. J. Exp. Med. 83:345-359 (1951); Barencamp et al., Infect. Immun. 60:1302-1313 (1992)). E. coli DH5α is a nonadherent laboratory strain that was originally obtained from Gibco BRL. E. coli strain BL2 1 (DE3) was a gift from F. W. Studier and contains a single copy of the T7 RNA polymerase gene under the control of the lac regulatory system (Studier, F. W., and B. A. Moffatt. 1986. Use of bacteriophage T7 RNA polymerase to direct high-level expression of cloned genes. J. Mol. Biol.189:113-130.). Plasmid pT7-7 was provided by S. Tabor and contains the T7 RNA polymerase promoter f10, a ribosome-bindingsite, and the translational start site for the T7 gene 10 protein upstream from a multiple cloning site (Tabor, S., and C. C. Richardson. 1985. A bacteriophage T7 RNA polymerase/promotersystem for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA. 82:1074-1078.). pUC 19 is a high-copy-number plasmid that has been previously described (Yanish-Perronet al., Gene 33:103-119(1985)). pDC400 is a pUC19 derivative that harbors the H. influenzae strain C54 surface fibril locus and is sufficient to promote in vitro adherence by laboratory strains of E. coli (St. Geme, J. W. III, and D. Cutter. 1995. Evidence that surface fibrils expressed by Haemophilus influenzae type b promote attachment to human epithelial cells. Mol. Microbiol. 15:77-85.). pHMW8-5 is a pT7-7 derivative that contains the H. influenzae strain 1I1 hia locus and also promotes adherence by nonadherent laboratory strains of E. coli (Barenkamp, S. J., and J. W. St. Geme, Ill. Identification of a second family of high molecular weight adhesion proteins expressed by nontypable Haemophilus influenzae. Mol. Microbiol., in press.). pHMW8-6 contains the H. influenzae hia locus interrupted by a kanamycin cassette (Barenkamp, S. J., and J. W. St. Geme, Ill. Identification of a second family of high molecular weight adhesion proteins expressed by nontypable Haemophilus influenzae. Mol. Microbiol., in press.). pUC4K served as the source of the kanamycin-resistancegene that was used as a probe in Southern analysis (Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 19:259-268.).

Culture conditions

H. influenzae strains were grown on chocolate agar supplemented with 1% Isovitale X, on brain heart infusion agar supplemented with hemin and NAD (BHI-DB agar), or in brain heart infusion broth supplemented with hemin and NAD (BHIs) (Anderson, P., R. B. Johnston, Jr., and D. H. Smith. 1972. Human serum activity against Haemophilus influenzae type b. J. Clin. Invest. 51:31-38.). These strains were stored at −80° C. in brain heart infusionbroth with 25% glycerol. E. coli strains were grown on Luria Bertani (LB) agar or in LB broth and were stored at −80° C. in LB broth with 50% glycerol. For H. influenzae, kanamycin was used in a concentration of 25 mg/ml. Antibiotic concentrations for E. coli included the following: ampicillin or carbenicillin 100 mg/ml and kanamycin 50 mg/ml.

Induction of plasmid-encoded proteins

To identify plasmid-encoded proteins, the bacteriophage T7 expression vector pT7-7 was employed and the relevant pT7-7 derivatives were transformed into E. coli BL21 (DE3). Activation of the T7 promoter was achieved by inducing expression of T7 RNA polymerase with isopropyl-b-D-thiogalactopyranoside (final concentration, 1 mM). After induction for 30 minutes at 37° C., rifampicin was added to a final concentration of 200 mg/ml. Thirty minutes later, 1 ml of culture was pulsed with 50 mCi of trans-[³⁵S]-label (ICN, Irvine, Calif.) for 5 minutes. Bacteria were harvested, and whole cell lysates were resuspended in Laemmli buffer for analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 7.5% acrylamide gels (Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London). 227:680-685.). Autoradiography was performed with Kodak XAR-5 film.

Recombinant DNA methods

DNA ligations, restriction endonuclease digestions, and gel electrophoresis were performed according to standard techniques (Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Plasmids were introduced into E. coli strains by either chemical transformation or electroporation, as described (Dower, W. J., J. F. Miller, and C. W. Ragsdale. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16:6127-6145., Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Transformation in H. influenzae was performed using the MIV method of Herriott et al. (Herriott, R. M., E. M. Meyer, and M. Vogt. 1970. Defined nongrowth media for stage II competence in Haemophilus influenzae. J. Bacteriol. 101:517-524.).

Adherence assays

Adherence assays were performed with tissue culture cells which were seeded into wells of 24-well tissue culture plates as previously described (St. Geme et al., Infect. Immun. 58:4036-4044(1991)). Adherence was measured after incubating bacteria with epithelial monolayers for 30 minutes as described (St. Geme, J. W. III, S. Falkow, and S. J. Barenkamp. 1993. High-molecular-weightproteins of nontypable Haemophilus influenzae mediate attachment to human epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 90:2875-2879.). Tissue culture cells included Chang epithelial cells (Wong-Kilbournederivative, clone 1-5c-4 (human conj unctiva))(ATCC CCL 20.2), KB cells (human oral epidermoid carcinoma) (ATCC CCL 17), HEp-2 cells (human laryngeal epidermoid carcinoma) (ATCC CCL 23), A549 cells (human lung carcinoma) (ATCC CCL 185), Intestine 407 cells (human embryonic intestine) (ATCC CCL 6), HeLa cells (human cervical epitheloid carcinoma) (ATCC CCL 2). ME- 180 cells (human cervical epidermoid carcinoma) (ATCC HTB 33), HEC-IB cells (human endometrium) (ATCC HTB 113), and CHO-K1 cells (Chinese hamster ovary) (ATCC CCL 61). Chang, KB, Intestine 407, HeLa, and HEC-IB cells were maintained in modified Eagle medium with Earle's salts and non-essential amino acids. HEp-2 cells were maintained in Dulbecco's modified Eagle medium, A549 cells and CHO-K1 cells in F12 medium (Ham), and ME-1 80 cells in McCoy5A medium. All media were supplemented with 10% heat-inactivated fetal bovine serum.

Southern analysis

Southern blotting was performed using high stringency conditions as previously described (St. Geme, J. W. III, and S. Falkow. 1991. Loss of capsule expression by Haemophilus influenzae type b results in enhanced adherence to and invasion of human cells. Infect. Immun. 59:1325-1333.).

Microscopy

Samples of epithelial cells with associated bacteria were stained with Giemsa stain and examined by light microscopy as described (St. Geme, J. W. III, and S. Falkow, S. 1990. Haemophilus influenzae adheres to and enters cultured human epithelial cells. Infect. Immun. 58:4036-4044.).

For negative-staining electron microscopy, bacteria were stained with 0.5% aqueous uranyl acetate (St. Geme, J. W. III, and S. Falkow. 1991. Loss of capsule expression by Haemophilus influenzae type b results in enhanced adherence to and invasion of human cells. Infect. Immun. 59:1325-1333.) and examined using a Zeiss 10A microscope.

The previous study indicated that laboratory E. coli strains harboring the plasmid pDC400 were capable of efficient attachment to cultured human epithelial cells (St. Geme, J. W. III, and D. Cutter. 1995. Evidence that surface fibrils expressed by Haemophilus influenzae type b promote attachment to human epithelial cells. Mol. Microbiol. 15:77-85.). Subcloning studies and transposon mutagenesis indicated that the relevant coding region of pDC400 was present within an 8.3 kb Xbal fragment(St. Geme, J. W. III, and D. Cutter. 1995. Evidence that surface fibrils expressed by Haemophilus influenzae type b promote attachment to human epithelial cells. Mol. Microbiol. 15:77-85.) (FIG. 7). To confirm this conclusion, in the present study this XbaI fragment was subcloned into pT7-7, generating plasmids designated pDC601 and pDC602, which contained the insert in opposite orientations (FIG. 7). As predicted, expression of these plasmids in E. coli DH5α was associated with a capacity for high level in vitro attachment (Table 1).

TABLE 1 Adherence to Chang conjunctival cells. Strain ADHERENCE (% inoculum)^(a) DH5α/pT7-7  0.4 ± 0.1 DH5α/pDC400 25.3 ± 1.2 DH5α/pDC601 54.3 ± 7.5 DH5α/pDC602 55.5 ± 4.3 C54b⁻p⁻ 98.7 ± 9.5 C54-HA1::kan^(b)  1.5 ± 0.2 C54-Tn400.23^(c)  3.3 ± 0.4 ^(a)Adherence was measured in a 30 minute assay and was calculated by dividing the number of adherent bacteria by the number of inoculated bacteria. Values are the mean ± SEM of measurements made in triplicate from representative experiments. ^(b)Strain C54-HA1::kan was constructed by transforming C54b⁻p⁻ with linearized pHMW8-6, which contains the HA1 gene with an intragenic kanamycin cassette. ^(c)Strain C54-Tn400.23 contains a mini-Tn10 kan element in the hsflocus (St. Geme et al., Mol. Microbiol. 15:77-85 (1995)).

To determine the direction of transcription and identify plasmid-encoded proteins, pDC601 and pDC602 were subsequently introduced into E. coli BL21(DE3), producing BL21(DE3)/pDC601 and BL21(DE3)/pDC602, respectively. As a negative control, pT7-7 was also transformed into BL2 I (DE3). The T7 promoter in these three strains was induced with IPTG, and induced proteins were detected using trans-[³⁵S]-label. As shown in FIG. 8, induction of BL21 (DE3)/pDC601 resulted in expression of a large protein over 200 kDa in size along with several slightly smaller proteins, which presumably represent degradation products. In contrast, when BL21 (DE3)/pDC602 and BL21 (DE3)/pT7-7 were induced, there was no expression of these proteins. This experiment indicated that the genetic material contained in the 8.3 kb XbaI fragment is transcribed from left to right as shown in FIG. 7 and suggested that a single long open reading frame may be present.

Nucleotide sequencing

Nucleotide sequence was determined using a Sequenase kit and double-stranded plasmid template. DNA fragments were subcloned into pUC 19 and sequenced along both strands by primer walking. DNA sequence analysis was performed using the Genetics Computer Group (GCG) software package from the University of Wisconsin (Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395.). Sequence similarity searches were carried out using the BLAST program of the National Center for Biotechnology Information (Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basis local alignment search tool. J. Mol. Biol. 215:403-410.).

Sequencing of the 8.3 kb Xbal fragment revealed a 7059 bp gene, which is designated for literature purposes as hsf for Hae6mophilus surface fibrils, and is referred to herein as HA2. This gene encodes a 2353-amino acid polypeptide, referred to as Hsf or HA2, with a calculated molecular mass of 243.8 kDa, which is similar in size to the observed protein species detected after induction of BL21(DE3)/pDC601. The HA2 gene has a GC content of 42.8%, somewhat greater than the published estimate of 38-39% for the whole genome (Fleischmann et al., 1995. Whole-genomerandom sequencing and assembly of Haemophilus influenzae Rd. Science. 269: 496-512., Kilian, M. 1976. A taxonomic study of the genus Haemophilus, with proposal of a new species. J. Gen. Microbiol. 93:9-62.). A putative ribosomal binding site with the sequence AAGGTA begins 13 base pairs upstream of the presumed initiation codon. A sequence similar to a rho-independent transcription terminator is present beginning 20 nucleotides beyond the stop codon and contains interrupted inverted repeats with the potential for forming a hairpin structure containing a loop of two bases and a stem of 11 bases. Of note, a string of 29 thymines spans the region from 149 to 121 nucleotides upstream of HA2.

Homology to Al4/HA1

The nontypable H. influenzae nonpilus protein HA1 protein (called Hia in the literature) promotes attachment to cultured human epithelial cells as outlined above. Comparison of the predicted amino acid sequence of A42 and the sequence of HA1 revealed 81% similarity and 72% identity overall. As depicted in FIG. 5 (SEQ ID NOS: 5 and 6), the two sequences are highly conserved at their N-terminal and C-terminal ends, and both contain a Walker box nucleotide-binding motif. Interestingly, HA1 is encoded by a 3.2 kb gene and is only 111052 5-kDa. In this context, it is noteworthy that three separate stretches of HA2 (corresponding to amino acids 174 to 608, 847 to 1291, and 1476 to 1914, respectively) show significant homology to the region of HA1 defined by amino acids 221 to 658 (FIG. 5). Table 2 summarizes the level of similarity and identity between these three stretches of HA2 and one another. The suggestion is that the larger size of HA2 may relate in part to the presence of a repeated domain which is present in single copy in HA1.

TABLE 2 Percent similarity and percent identity between HA2 repeats. Percent Similarity/Percent Identity HA2 174-608^(a) HA2 847-1291^(a) HA2 1476-1914^(a) HA2 174-608 * 65/53 76/60 HA2 847-1291 * 70/56 HA2 1476-1914 * ^(a)Numbers correspond to amino acid residue positions in the full-length HA2 (Hsf) protein.

To evaluate whether HA1 and HA2 are alleles of the same locus, a series of Southern blots were performed. Samples of chromosomal DNA from strains C54 and 11 were subjected to digestion with BglII, ClaI and either PstI or XbaI. Resulting DNA fragments were separated by agarose electrophoresis and transferred bidirectionally to nitrocellulose membranes. One membrane was probed with a 3.3 kb internal fragment of the HA2 gene (FIG. 7), and the other membrane was probed with a 1.6 kb intragenic fragment of the HA1 gene. As shown in FIG. 9, both probes recognized exactly the same chromosomal fragments.

To obtain additional evidence that the HA2 and HA1 genes are homologs, the inactivation of HA2 by transformation of H. influenzae strain C54bp with insertionally inactivated HA1 was attempted. The plasmid pHMW8-6 (Barenkamp, S. J., and J. W. St. Geme, III. Identification of a second family of high molecular weight adhesion proteins expressed by nontypable Haemophilus influenzae. Mol. Microbiol., in press.), which contains the HA1 gene with an intragenic kanamycin cassette, was linearized with NdeI and introduced into competent C54. Southern hybridization confirmed insertion of the kanamycin cassette into HA2 (not shown). Furthermore examination of the C54 mutant by negative staining transmission electron microscopy revealed the loss of surface fibrils (not shown). Consistent with these findings, the mutant strain demonstrated minimal attachment to Chang conjunctival cells (Table 1).

In additional experiments, the cellular binding specificities conferred by the HA2 and HA1 proteins were compared. As shown in FIG. 10, DH5α/pDC601 (expressing HA2) demonstrated high level attachment to Chang cells, KB cells. HeLa cells, and Intestine407 cells, moderate level attachment to HEp-2 cells, and minimal attachment to HEC-IB cells, ME-180 cells, and CHO-K1 cells. DH5α harboring pHMW8-5 (expressing HA1) showed virtually the same pattern of attachment. Giemsa staining and subsequent examination by light microscopy confirmed these viable count adherence assay results.

Homology to other bacterial extracellular proteins

A protein sequence similarity search was performed with the HA2 predicted amino acid sequence (SEQ ID NO:7) using the BLAST network service of the National Center for Biotechnology Information (Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basis local alignment search tool. J. Mol. Biol. 215:403-410.). This search revealed low-level sequence similarity to a series of other bacterial adherence factors, including HMW1 (SEQ ID NO:9) and HMW2 (SEQ ID NO:10) (the proteins previously identified as being important adhesins in HA1-deficient nontypable H. influenzae strains; (St. Geme, J. W. III, S. Falkow, and S. J. Barenkamp. 1993. High-molecular-weight proteins of nontypable Haemophilus influenzae mediate attachment to human epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 90:2875-2879.), AIDA-I (SEQ ID NO:11) (an adhesion protein expressed by some diarrheagenic E. coli strains: Benz, I., and M. A. Schmidt. 1992. AIDA-I (SEQ ID NO:11). the adhesin involved in diffuse adherence of the diarrhoeagenic Escherichia coli strain 2787 (0126:H27), is synthesized via a precursor molecule. Mol. Microbiol. 6:1539-1546.),and Tsh (SEQ ID NO:12) (a hemagglutinin produced by an avian pathogenic E. coli strain; Provence, D. and R. Curtiss III1994. Isolation and characterization of a gene involved in hemagglutination by an avian pathogenic Escherichia coli strain. Infect Immun. 62:1369-1380.). In addition. HA2 (SEQ ID NO:7) showed homology to SepA (SEQ ID NO:13), a Shigella flexneri secreted protein that appears to play a role in tissue invasion (Benjelloun-Touimi, Z. P. J. Sansonetti, and C. Parsot. 1995. SepA (SEQ ID NO:13). the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion. Mol. Microbiol. 17:123-135.). Alignment of HA2 (SEQ ID NO:7) with HMW1, (SEQ ID NO:9) HMW2 (SEQ ID NO:10), AIDA-I (SEQ ID NO:11), Tsh (SEQ ID NO:12), and SepA (SEQ ID NO:13) revealed a highly conserved N-terminal domain (FIG. 11). In AIDA-I (SEQ ID NO:11), Tsh (SEQ ID NO:12), and SepA (SEQ ID NO:13), this N-terminal extremity precedes a typical procaryotic signal sequence (Benjelloun-Touimi.Z., P. J. Sansonetti, and C. Parsot. 1995. SepA (SEQ ID NO:13), the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion. Mol. Microbiol. 17:123-135.). Similarly, in HA2 this conserved domain precedes a 26 amino acid segment that is characterized by a positively charged region, followed by a string of hydrophobic residues, and then alanine-glutamine-alanine.

Presence of an HA2 homolog in other encapsulated and nonencapsulated strains

Previous work demonstrated that an HA2 homolog is present in H. influenzae type b strains M42 and Eagan (St. Geme, J. W. III, and D. Cutter. 1995. Evidence that surface fibrils expressed by Haemophilus influenzae type b promote attachment to human epithelial cells. Mol. Microbiol. 15:77-85.). To define the extent to which the HA2 locus is shared by other type b strains, a panel of evolutionarily diverse type b isolates by Southern analysis were examined. Among these strains were six belonging to phylogenic division I and four belonging to phylogenic division II (Musser, J. M., J. S. Kroll, E. R. Moxon, and R. K. Selander. 1988. Evolutionary genetics of the encapsulated strains of Haemophilus influenzae. Proc. Natl. Acad. Sci. U.S.A. 85:7758-7762.). Chromosomal DNA was digested with BglII and then probed with the intragenic 3.3 kb fragment of the HA2 gene. As shown in FIG. 12, all 10 strains showed hybridization. The universal presence among H. influenzae type b raised the question of the prevalence of this locus in other non-type b encapsulated H. influenzae. Southern analysis of a series of type a, c, d, e, and f isolates again demonstrated a homolog in all cases (FIG. 13).

Recently Fleischmann et al. (Fleischmann R. D., et al., 1995. Whole-genomerandom sequencing and assembly of Haemophilus influenzae Rd. Science. 269: 496-512.) reported the genome sequence of H. influenzae strain Rd, which was one of the two serotype d strains examined by Southern analysis. In accord with the Southern blotting results, search of the Rd genome revealed an open reading frame with striking sequence similarity to HA2. The Rd gene is 894 nucleotides in length and is predicted to encode a protein of 298 amino acids. Overall, the Rd locus is 70% identical to the C54 HA2 gene, and the Rd derived amino acid sequence is 62% identical and 75% similar to C54 HA2. Interestingly, the Rd open reading frame appears to be truncated due to a “premature” stop codon.

Previous experiments revealed that 13 of 15 nontypable strains lacking an HMW 1 /HMW2-related protein had evidence of an HA1 homolog (Barenkamp, S. J., and J. W. St. Geme, III. Identification of a second family of high molecular weight adhesion proteins expressed by nontypable Haemophilus influenzae. Mol. Microbiol., in press.). Consistent with the demonstration that HA2 and HA1 are homologous, Southern analysis of these 15 strains, probing with the 3.3 kb fragment of hsf, demonstrated hybridization in 12 of the same 13 (not shown).

Chromosomal location of the HA2 locus

In earlier work, the HA1 locus in nontypable strain 11 was found to be flanked upstream by an open reading frame with significant homology to E. coli exoribonuclease II (Barenkamp, S. J., and J. W. St. Geme, III. Identification of a second family of high molecular weight adhesion proteins expressed by nontypable Haemophilus influenzae. Mol. Microbiol., in press.). Similarly, the HA2 locus in strain C54 likewise is flanked on the 5′ side by an open reading frame with similarity to E. coli exonuclease II. This gene terminates 357 base pairs before the HA2 start codon and encodes a protein with a predicted amino acid sequence that is 61% similar and 33% identical at its C-terminal end to exoribonuclease II. Of note, the Rd HA2 homolog is also flanked upstream by the exoribonuclease II locus.

EXAMPLE 3 Cloning of HA3

Recombinant phage containing the nontypable Haemophilus strain 32 HA3 gene were isolated and characterized using methods modified slightly from those described previously (Barenkamp and St. Geme, Molecular Microbiology 1996, in press). In brief, chromosomal DNA from strain 32 was prepared by a modification of the method of Marmur (Marmur, 1961). Sau3A partial restriction digests of the DNA were prepared fractionated on 0.7% agarose gels. Fractions containing DNA fragments in the 9- to 20- kbp range were pooled, and a library was prepared by ligation into λEMBL3 arms. Ligation mixtures were packaged in vitro with Gigapack® (Stratagene, La Jolla, Calif.) and plate amplified in a P2 lysogen of E. coli LE392.

Lambda plaque screening was performed using a mixture of three PCR products derived from strain 32 chromosomal DNA. These PCR products were amplified using primer pairs previously shown to amplify DNA segments at the 5′ end of the strain 11 HA1 gene. The primers were as follows:

Primer designation strand sequence 44P positive CCG TGC TTG CCC AAC ACG CTT (SEQ ID NO:16) 64P positive GCT GCC ACC TTG CAC AAC AAC (SEQ ID NO:17) 93G-2 positive CTT TCA ATG CCA GAA AGT AGG (SEQ ID NO:18) 18T-1 negative CTT CAA CCG TTG CGG ACA ACA (SEQ ID NO:19)

Each of the positive strand primers was used with the single negative strand primer to generate the three fragments used for probing the library.

The PCR products generated from strain I 11 and strain 32 chromosomal DNA were identical in size, suggesing that the nucleotide sequences of these chromosomal regions were similar in the two strains. Plaque screening was performed using standard methodology (Berger and Kimmel, 1987) at high stringency: final wash conditions were 65C for 1 hour in buffer containing 2XSSC and 1% SDS. Positive plaques were identified by autoradiography, plaque purified and phage DNA was purified by standard methods. The same primer pairs used to generate the screening probes were then used to localize the HA3 gene by amplifying various restriction fragments derived from the phage DNA. Once localized, the strain 32 HA3 gene and flanking DNA were sequenced using standard methods.

In order to construct strain 32 isogenic Haemophilus influenzae mutants deficient in expression of the HA3 gene, bacteria were made competent using the MIV (Herriott et al. 1970) and were transformed with linearized pHMW8-6, selecting for kanamycin resistance. Allelic exchange was confirmed by Southern analysis. The mutants that no longer expressed HA3 exhibited a marked decrease in binding to Chang epithelial cells, using the methods outlined above (data not shown).

Expression in non-adherent strains of E. coli did not result in adherence, although it has not been confirmed that the protein was actually expressed.

19 3294 base pairs nucleic acid unknown unknown DNA unknown 1 ATGAACAAAA TTTTTAACGT TATTTGGAAT GTTGTGACTC AAACTTGGGT TGTCGTATCT 60 GAACTCACTC GCACCCACAC CAAATGCGCC TCCGCCACCG TGGCGGTTGC CGTATTGGCA 120 ACCCTGTTGT CCGCAACGGT TGAGGCGAAC AACAATACTC CTGTTACGAA TAAGTTGAAG 180 GCTTATGGCG ATGCGAATTT TAATTTCACT AATAATTCGA TAGCAGATGC AGAAAAACAA 240 GTTCAAGAGG CTTATAAAGG TTTATTAAAT CTAAATGAAA AAAATGCGAG TGATAAACTG 300 TTGGTGGAGG ACAATACTGC GGCGACCGTA GGCAATTTGC GTAAATTGGG CTGGGTATTG 360 TCTAGCAAAA ACGGCACAAG GAACGAGAAA AGCCAACAAG TCAAACATGC GGATGAAGTG 420 TTGTTTGAAG GCAAAGGCGG TGTGCAGGTT ACTTCCACCT CTGAAAACGG CAAACACACC 480 ATTACCTTTG CTTTAGCGAA AGACCTTGGT GTGAAAACTG CGACTGTGAG TGATACCTTA 540 ACGATTGGCG GTGGTGCTGC TGCAGGTGCT ACAACAACAC CGAAAGTGAA TGTAACTAGT 600 ACAACTGATG GCTTGAAGTT CGCTAAAGAT GCTGCGGGTG CTAATGGCGA TACTACGGTT 660 CACTTGAATG GTATTGGTTC AACCTTGACA GACACGCTTG TGGGTTCTCC TGCTACTCAT 720 ATTGACGGAG GAGATCAAAG TACGCATTAC ACTCGTGCAG CAAGTATCAA GGATGTCTTG 780 AATGCGGGTT GGAATATCAA GGGTGTTAAA GCTGGCTCAA CAACTGGTCA ATCAGAAAAT 840 GTCGATTTTG TTCATACTTA CGATACTGTT GAGTTCTTGA GTGCGGATAC AGAGACCACG 900 ACTGTTACTG TAGATAGCAA AGAAAACGGT AAGAGAACCG AAGTTAAAAT CGGTGCGAAG 960 ACTTCTGTTA TCAAAGAAAA AGACGGTAAG TTATTTACTG GAAAAGCTAA CAAAGAGACA 1020 AATAAAGTTG ATGGTGCTAA CGCGACTGAA GATGCAGACG AAGGCAAAGG CTTAGTGACT 1080 GCGAAAGATG TGATTGACGC AGTGAATAAG ACTGGTTGGA GAATTAAAAC AACCGATGCT 1140 AATGGTCAAA ATGGCGACTT CGCAACTGTT GCATCAGGCA CAAATGTAAC CTTTGCTAGT 1200 GGTAATGGTA CAACTGCGAC TGTAACTAAT GGCACCGATG GTATTACCGT TAAGTATGAT 1260 GCGAAAGTTG GCGACGGCTT AAAACTAGAT GGCGATAAAA TCGCTGCAGA TACGACCGCA 1320 CTTACTGTGA ATGATGGTAA GAACGCTAAT AATCCGAAAG GTAAAGTGGC TGATGTTGCT 1380 TCAACTGACG AGAAGAAATT GGTTACAGCA AAAGGTTTAG TAACAGCCTT AAACAGTCTA 1440 AGCTGGACTA CAACTGCTGC TGAGGCGGAC GGTGGTACGC TTGATGGAAA TGCAAGTGAG 1500 CAAGAAGTTA AAGCGGGCGA TAAAGTAACC TTTAAAGCAG GCAAGAACTT AAAAGTGAAA 1560 CAAGAGGGTG CGAACTTTAC TTATTCACTG CAAGATGCTT TAACAGGCTT AACGAGCATT 1620 ACTTTAGGTA CAGGAAATAA TGGTGCGAAA ACTGAAATCA ACAAAGACGG CTTAACCATC 1680 ACACCAGCAA ATGGTGCGGG TGCAAATAAT GCAAACACCA TCAGCGTAAC CAAAGACGGC 1740 ATTAGTGCGG GCGGTCAGTC GGTTAAAAAC GTTGTGAGCG GACTGAAGAA ATTTGGTGAT 1800 GCGAATTTCG ATCCGCTGAC TAGCTCCGCC GACAACTTAA CGAAACAAAA TGACGATGCC 1860 TATAAAGGCT TGACCAATTT GGATGAAAAA GGTACAGACA AGCAAACTCC AGTTGTTGCC 1920 GACAATACCG CCGCAACCGT GGGCGATTTG CGCGGCTTGG GCTGGGTCAT TTCTGCGGAC 1980 AAAACCACAG GCGGCTCAAC GGAATATCAC GATCAAGTTC GGAATGCGAA CGAAGTGAAA 2040 TTCAAAAGCG GCAACGGTAT CAATGTTTCC GGTAAAACGG TCAACGGTAG GCGTGAAATT 2100 ACTTTTGAAT TGGCTAAAGG TGAAGTGGTT AAATCGAATG AATTTACCGT CAAAGAAACC 2160 AATGGAAAGG AAACGAGCCT GGTTAAAGTT GGCGATAAAT ATTACAGCAA AGAGGATATT 2220 GACTTAACAA CAGGTCAGCC TAAATTAAAA GATGGCAATA CAGTTGCTGC GAAATATCAA 2280 GATAAAGGTG GCAAAGTCGT TTCTGTAACG GATAATACTG AAGCTACCAT AACCAACAAA 2340 GGTTCTGGCT ATGTAACAGG TAACCAAGTG GCAGATGCGA TTGCGAAATC AGGCTTTGAG 2400 CTTGGCTTGG CTGATGAAGC TGATGCGAAA CGGGCGTTTG ATGATAAGAC AAAAGCCTTA 2460 TCTGCTGGTA CAACGGAAAT TGTAAATGCC CACGATAAAG TCCGTTTTGC TAATGGTTTA 2520 AATACCAAAG TGAGCGCGGC AACGGTGGAA AGCACCGATG CAAACGGCGA TAAAGTGACC 2580 ACAACCTTTG TGAAAACCGA TGTGGAATTG CCTTTAACGC AAATCTACAA TACCGATGCA 2640 AACGGTAAGA AAATCACTAA AGTTGTCAAA GATGGGCAAA CTAAATGGTA TGAACTGAAT 2700 GCTGACGGTA CGGCTGATAT GACCAAAGAA GTTACCCTCG GTAACGTGGA TTCAGACGGC 2760 AAGAAAGTTG TGAAAGACAA CGATGGCAAG TGGTATCACG CCAAAGCTGA CGGTACTGCG 2820 GATAAAACCA AAGGCGAAGT GAGCAATGAT AAAGTTTCTA CCGATGAAAA ACACGTTGTC 2880 AGCCTTGATC CAAATGATCA ATCAAAAGGT AAAGGTGTCG TGATTGACAA TGTGGCTAAT 2940 GGCGATATTT CTGCCACTTC CACCGATGCG ATTAACGGAA GTCAGTTGTA TGCTGTGGCA 3000 AAAGGGGTAA CAAACCTTGC TGGACAAGTG AATAATCTTG AGGGCAAAGT GAATAAAGTG 3060 GGCAAACGTG CAGATGCAGG TACAGCAAGT GCATTAGCGG CTTCACAGTT ACCACAAGCC 3120 ACTATGCCAG GTAAATCAAT GGTTGCTATT GCGGGAAGTA GTTATCAAGG TCAAAATGGT 3180 TTAGCTATCG GGGTATCAAG AATTTCCGAT AATGGCAAAG TGATTATTCG CTTGTCAGGC 3240 ACAACCAATA GTCAAGGTAA AACAGGCGTT GCAGCAGGTG TTGGTTACCA GTGG 3294 1098 amino acids amino acid unknown unknown protein unknown 2 Met Asn Lys Ile Phe Asn Val Ile Trp Asn Val Val Thr Gln Thr Trp 1 5 10 15 Val Val Val Ser Glu Leu Thr Arg Thr His Thr Lys Cys Ala Ser Ala 20 25 30 Thr Val Ala Val Ala Val Leu Ala Thr Leu Leu Ser Ala Thr Val Glu 35 40 45 Ala Asn Asn Asn Thr Pro Val Thr Asn Lys Leu Lys Ala Tyr Gly Asp 50 55 60 Ala Asn Phe Asn Phe Thr Asn Asn Ser Ile Ala Asp Ala Glu Lys Gln 65 70 75 80 Val Gln Glu Ala Tyr Lys Gly Leu Leu Asn Leu Asn Glu Lys Asn Ala 85 90 95 Ser Asp Lys Leu Leu Val Glu Asp Asn Thr Ala Ala Thr Val Gly Asn 100 105 110 Leu Arg Lys Leu Gly Trp Val Leu Ser Ser Lys Asn Gly Thr Arg Asn 115 120 125 Glu Lys Ser Gln Gln Val Lys His Ala Asp Glu Val Leu Phe Glu Gly 130 135 140 Lys Gly Gly Val Gln Val Thr Ser Thr Ser Glu Asn Gly Lys His Thr 145 150 155 160 Ile Thr Phe Ala Leu Ala Lys Asp Leu Gly Val Lys Thr Ala Thr Val 165 170 175 Ser Asp Thr Leu Thr Ile Gly Gly Gly Ala Ala Ala Gly Ala Thr Thr 180 185 190 Thr Pro Lys Val Asn Val Thr Ser Thr Thr Asp Gly Leu Lys Phe Ala 195 200 205 Lys Asp Ala Ala Gly Ala Asn Gly Asp Thr Thr Val His Leu Asn Gly 210 215 220 Ile Gly Ser Thr Leu Thr Asp Thr Leu Val Gly Ser Pro Ala Thr His 225 230 235 240 Ile Asp Gly Gly Asp Gln Ser Thr His Tyr Thr Arg Ala Ala Ser Ile 245 250 255 Lys Asp Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Ala Gly 260 265 270 Ser Thr Thr Gly Gln Ser Glu Asn Val Asp Phe Val His Thr Tyr Asp 275 280 285 Thr Val Glu Phe Leu Ser Ala Asp Thr Glu Thr Thr Thr Val Thr Val 290 295 300 Asp Ser Lys Glu Asn Gly Lys Arg Thr Glu Val Lys Ile Gly Ala Lys 305 310 315 320 Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu Phe Thr Gly Lys Ala 325 330 335 Asn Lys Glu Thr Asn Lys Val Asp Gly Ala Asn Ala Thr Glu Asp Ala 340 345 350 Asp Glu Gly Lys Gly Leu Val Thr Ala Lys Asp Val Ile Asp Ala Val 355 360 365 Asn Lys Thr Gly Trp Arg Ile Lys Thr Thr Asp Ala Asn Gly Gln Asn 370 375 380 Gly Asp Phe Ala Thr Val Ala Ser Gly Thr Asn Val Thr Phe Ala Ser 385 390 395 400 Gly Asn Gly Thr Thr Ala Thr Val Thr Asn Gly Thr Asp Gly Ile Thr 405 410 415 Val Lys Tyr Asp Ala Lys Val Gly Asp Gly Leu Lys Leu Asp Gly Asp 420 425 430 Lys Ile Ala Ala Asp Thr Thr Ala Leu Thr Val Asn Asp Gly Lys Asn 435 440 445 Ala Asn Asn Pro Lys Gly Lys Val Ala Asp Val Ala Ser Thr Asp Glu 450 455 460 Lys Lys Leu Val Thr Ala Lys Gly Leu Val Thr Ala Leu Asn Ser Leu 465 470 475 480 Ser Trp Thr Thr Thr Ala Ala Glu Ala Asp Gly Gly Thr Leu Asp Gly 485 490 495 Asn Ala Ser Glu Gln Glu Val Lys Ala Gly Asp Lys Val Thr Phe Lys 500 505 510 Ala Gly Lys Asn Leu Lys Val Lys Gln Glu Gly Ala Asn Phe Thr Tyr 515 520 525 Ser Leu Gln Asp Ala Leu Thr Gly Leu Thr Ser Ile Thr Leu Gly Thr 530 535 540 Gly Asn Asn Gly Ala Lys Thr Glu Ile Asn Lys Asp Gly Leu Thr Ile 545 550 555 560 Thr Pro Ala Asn Gly Ala Gly Ala Asn Asn Ala Asn Thr Ile Ser Val 565 570 575 Thr Lys Asp Gly Ile Ser Ala Gly Gly Gln Ser Val Lys Asn Val Val 580 585 590 Ser Gly Leu Lys Lys Phe Gly Asp Ala Asn Phe Asp Pro Leu Thr Ser 595 600 605 Ser Ala Asp Asn Leu Thr Lys Gln Asn Asp Asp Ala Tyr Lys Gly Leu 610 615 620 Thr Asn Leu Asp Glu Lys Gly Thr Asp Lys Gln Thr Pro Val Val Ala 625 630 635 640 Asp Asn Thr Ala Ala Thr Val Gly Asp Leu Arg Gly Leu Gly Trp Val 645 650 655 Ile Ser Ala Asp Lys Thr Thr Gly Gly Ser Thr Glu Tyr His Asp Gln 660 665 670 Val Arg Asn Ala Asn Glu Val Lys Phe Lys Ser Gly Asn Gly Ile Asn 675 680 685 Val Ser Gly Lys Thr Val Asn Gly Arg Arg Glu Ile Thr Phe Glu Leu 690 695 700 Ala Lys Gly Glu Val Val Lys Ser Asn Glu Phe Thr Val Lys Glu Thr 705 710 715 720 Asn Gly Lys Glu Thr Ser Leu Val Lys Val Gly Asp Lys Tyr Tyr Ser 725 730 735 Lys Glu Asp Ile Asp Leu Thr Thr Gly Gln Pro Lys Leu Lys Asp Gly 740 745 750 Asn Thr Val Ala Ala Lys Tyr Gln Asp Lys Gly Gly Lys Val Val Ser 755 760 765 Val Thr Asp Asn Thr Glu Ala Thr Ile Thr Asn Lys Gly Ser Gly Tyr 770 775 780 Val Thr Gly Asn Gln Val Ala Asp Ala Ile Ala Lys Ser Gly Phe Glu 785 790 795 800 Leu Gly Leu Ala Asp Glu Ala Asp Ala Lys Arg Ala Phe Asp Asp Lys 805 810 815 Thr Lys Ala Leu Ser Ala Gly Thr Thr Glu Ile Val Asn Ala His Asp 820 825 830 Lys Val Arg Phe Ala Asn Gly Leu Asn Thr Lys Val Ser Ala Ala Thr 835 840 845 Val Glu Ser Thr Asp Ala Asn Gly Asp Lys Val Thr Thr Thr Phe Val 850 855 860 Lys Thr Asp Val Glu Leu Pro Leu Thr Gln Ile Tyr Asn Thr Asp Ala 865 870 875 880 Asn Gly Lys Lys Ile Thr Lys Val Val Lys Asp Gly Gln Thr Lys Trp 885 890 895 Tyr Glu Leu Asn Ala Asp Gly Thr Ala Asp Met Thr Lys Glu Val Thr 900 905 910 Leu Gly Asn Val Asp Ser Asp Gly Lys Lys Val Val Lys Asp Asn Asp 915 920 925 Gly Lys Trp Tyr His Ala Lys Ala Asp Gly Thr Ala Asp Lys Thr Lys 930 935 940 Gly Glu Val Ser Asn Asp Lys Val Ser Thr Asp Glu Lys His Val Val 945 950 955 960 Ser Leu Asp Pro Asn Asp Gln Ser Lys Gly Lys Gly Val Val Ile Asp 965 970 975 Asn Val Ala Asn Gly Asp Ile Ser Ala Thr Ser Thr Asp Ala Ile Asn 980 985 990 Gly Ser Gln Leu Tyr Ala Val Ala Lys Gly Val Thr Asn Leu Ala Gly 995 1000 1005 Gln Val Asn Asn Leu Glu Gly Lys Val Asn Lys Val Gly Lys Arg Ala 1010 1015 1020 Asp Ala Gly Thr Ala Ser Ala Leu Ala Ala Ser Gln Leu Pro Gln Ala 1025 1030 1035 1040 Thr Met Pro Gly Lys Ser Met Val Ala Ile Ala Gly Ser Ser Tyr Gln 1045 1050 1055 Gly Gln Asn Gly Leu Ala Ile Gly Val Ser Arg Ile Ser Asp Asn Gly 1060 1065 1070 Lys Val Ile Ile Arg Leu Ser Gly Thr Thr Asn Ser Gln Gly Lys Thr 1075 1080 1085 Gly Val Ala Ala Gly Val Gly Tyr Gln Trp 1090 1095 7291 base pairs nucleic acid unknown unknown DNA unknown CDS 163..7221 3 TTTNTTTTTC TTATTTTTTT TTTTTTTTTT TTTTTTTTTT TTGAGGCTAA ACTTTTNGNA 60 AAATATCACT TTTTTATTCT CCAAATATAG AATAGAATAC GCACGATTTC ACTAAGAAAA 120 GTATATTTAT CATTAATTTT ATTAAATATA AGGTAAATAA AA ATG AAC AAA ATT 174 Met Asn Lys Ile 1 TTT AAC GTT ATT TGG AAT GTT ATG ACT CAA ACT TGG GTT GTC GTA TCT 222 Phe Asn Val Ile Trp Asn Val Met Thr Gln Thr Trp Val Val Val Ser 5 10 15 20 GAA CTC ACT CGC ACC CAC ACC AAA CGC GCC TCC GCA ACC GTG GAG ACC 270 Glu Leu Thr Arg Thr His Thr Lys Arg Ala Ser Ala Thr Val Glu Thr 25 30 35 GCC GTA TTG GCG ACA CTG TTG TTT GCA ACG GTT CAG GCG AAT GCT ACC 318 Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln Ala Asn Ala Thr 40 45 50 GAT GAA GAT GAA GAG TTA GAC CCC GTA GTA CGC ACT GCT CCC GTG TTG 366 Asp Glu Asp Glu Glu Leu Asp Pro Val Val Arg Thr Ala Pro Val Leu 55 60 65 AGC TTC CAT TCC GAT AAA GAA GGC ACG GGA GAA AAA GAA GTT ACA GAA 414 Ser Phe His Ser Asp Lys Glu Gly Thr Gly Glu Lys Glu Val Thr Glu 70 75 80 AAT TCA AAT TGG GGA ATA TAT TTC GAC AAT AAA GGA GTA CTA AAA GCC 462 Asn Ser Asn Trp Gly Ile Tyr Phe Asp Asn Lys Gly Val Leu Lys Ala 85 90 95 100 GGA GCA ATC ACC CTC AAA GCC GGC GAC AAC CTG AAA ATC AAA CAA AAC 510 Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu Lys Ile Lys Gln Asn 105 110 115 ACC GAT GAA AGC ACC AAT GCC AGT AGC TTC ACC TAC TCG CTG AAA AAA 558 Thr Asp Glu Ser Thr Asn Ala Ser Ser Phe Thr Tyr Ser Leu Lys Lys 120 125 130 GAC CTC ACA GAT CTG ACC AGT GTT GCA ACT GAA AAA TTA TCG TTT GGC 606 Asp Leu Thr Asp Leu Thr Ser Val Ala Thr Glu Lys Leu Ser Phe Gly 135 140 145 GCA AAC GGC GAT AAA GTT GAT ATT ACC AGT GAT GCA AAT GGC TTG AAA 654 Ala Asn Gly Asp Lys Val Asp Ile Thr Ser Asp Ala Asn Gly Leu Lys 150 155 160 TTG GCG AAA ACA GGT AAC GGA AAT GTT CAT TTG AAT GGT TTG GAT TCA 702 Leu Ala Lys Thr Gly Asn Gly Asn Val His Leu Asn Gly Leu Asp Ser 165 170 175 180 ACT TTG CCT GAT GCG GTA ACG AAT ACA GGT GTG TTA AGT TCA TCA AGT 750 Thr Leu Pro Asp Ala Val Thr Asn Thr Gly Val Leu Ser Ser Ser Ser 185 190 195 TTT ACA CCT AAT GAT GTT GAA AAA ACA AGA GCT GCA ACT GTT AAA GAT 798 Phe Thr Pro Asn Asp Val Glu Lys Thr Arg Ala Ala Thr Val Lys Asp 200 205 210 GTT TTA AAT GCA GGT TGG AAC ATT AAA GGT GCT AAA ACT GCT GGA GGT 846 Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Ala Lys Thr Ala Gly Gly 215 220 225 AAT GTT GAG AGT GTT GAT TTA GTG TCC GCT TAT AAT AAT GTT GAA TTT 894 Asn Val Glu Ser Val Asp Leu Val Ser Ala Tyr Asn Asn Val Glu Phe 230 235 240 ATT ACA GGC GAT AAA AAC ACG CTT GAT GTT GTA TTA ACA GCT AAA GAA 942 Ile Thr Gly Asp Lys Asn Thr Leu Asp Val Val Leu Thr Ala Lys Glu 245 250 255 260 AAC GGT AAA ACA ACC GAA GTG AAA TTC ACA CCG AAA ACC TCT GTT ATC 990 Asn Gly Lys Thr Thr Glu Val Lys Phe Thr Pro Lys Thr Ser Val Ile 265 270 275 AAA GAA AAA GAC GGT AAG TTA TTT ACT GGA AAA GAG AAT AAC GAC ACA 1038 Lys Glu Lys Asp Gly Lys Leu Phe Thr Gly Lys Glu Asn Asn Asp Thr 280 285 290 AAT AAA GTT ACA AGT AAC ACG GCG ACT GAT AAT ACA GAT GAG GGT AAT 1086 Asn Lys Val Thr Ser Asn Thr Ala Thr Asp Asn Thr Asp Glu Gly Asn 295 300 305 GGC TTA GTC ACT GCA AAA GCT GTG ATT GAT GCT GTG AAC AAG GCT GGT 1134 Gly Leu Val Thr Ala Lys Ala Val Ile Asp Ala Val Asn Lys Ala Gly 310 315 320 TGG AGA GTT AAA ACA ACT ACT GCT AAT GGT CAA AAT GGC GAC TTC GCA 1182 Trp Arg Val Lys Thr Thr Thr Ala Asn Gly Gln Asn Gly Asp Phe Ala 325 330 335 340 ACT GTT GCG TCA GGC ACA AAT GTA ACC TTT GAA AGT GGC GAT GGT ACA 1230 Thr Val Ala Ser Gly Thr Asn Val Thr Phe Glu Ser Gly Asp Gly Thr 345 350 355 ACA GCG TCA GTA ACT AAA GAT ACT AAC GGC AAT GGC ATC ACT GTT AAG 1278 Thr Ala Ser Val Thr Lys Asp Thr Asn Gly Asn Gly Ile Thr Val Lys 360 365 370 TAC GAC GCG AAA GTT GGC GAC GGC TTG AAA TTT GAT AGC GAT AAA AAA 1326 Tyr Asp Ala Lys Val Gly Asp Gly Leu Lys Phe Asp Ser Asp Lys Lys 375 380 385 ATC GTT GCA GAT ACG ACC GCA CTT ACT GTG ACA GGT GGT AAG GTA GCT 1374 Ile Val Ala Asp Thr Thr Ala Leu Thr Val Thr Gly Gly Lys Val Ala 390 395 400 GAA ATT GCT AAA GAA GAT GAC AAG AAA AAA CTT GTT AAT GCA GGC GAT 1422 Glu Ile Ala Lys Glu Asp Asp Lys Lys Lys Leu Val Asn Ala Gly Asp 405 410 415 420 TTG GTA ACA GCT TTA GGT AAT CTA AGT TGG AAA GCA AAA GCT GAG GCT 1470 Leu Val Thr Ala Leu Gly Asn Leu Ser Trp Lys Ala Lys Ala Glu Ala 425 430 435 GAT ACT GAT GGT GCG CTT GAG GGG ATT TCA AAA GAC CAA GAA GTC AAA 1518 Asp Thr Asp Gly Ala Leu Glu Gly Ile Ser Lys Asp Gln Glu Val Lys 440 445 450 GCA GGC GAA ACG GTA ACC TTT AAA GCG GGC AAG AAC TTA AAA GTG AAA 1566 Ala Gly Glu Thr Val Thr Phe Lys Ala Gly Lys Asn Leu Lys Val Lys 455 460 465 CAG GAT GGT GCG AAC TTT ACT TAT TCA CTG CAA GAT GCT TTA ACG GGT 1614 Gln Asp Gly Ala Asn Phe Thr Tyr Ser Leu Gln Asp Ala Leu Thr Gly 470 475 480 TTA ACG AGC ATT ACT TTA GGT GGT ACA ACT AAT GGC GGA AAT GAT GCG 1662 Leu Thr Ser Ile Thr Leu Gly Gly Thr Thr Asn Gly Gly Asn Asp Ala 485 490 495 500 AAA ACC GTC ATC AAC AAA GAC GGT TTA ACC ATC ACG CCA GCA GGT AAT 1710 Lys Thr Val Ile Asn Lys Asp Gly Leu Thr Ile Thr Pro Ala Gly Asn 505 510 515 GGC GGT ACG ACA GGT ACA AAC ACC ATC AGC GTA ACC AAA GAT GGC ATT 1758 Gly Gly Thr Thr Gly Thr Asn Thr Ile Ser Val Thr Lys Asp Gly Ile 520 525 530 AAA GCA GGT AAT AAA GCT ATT ACT AAT GTT GCG AGT GGT TTA AGA GCT 1806 Lys Ala Gly Asn Lys Ala Ile Thr Asn Val Ala Ser Gly Leu Arg Ala 535 540 545 TAT GAC GAT GCG AAT TTT GAT GTT TTA AAT AAC TCT GCA ACT GAT TTA 1854 Tyr Asp Asp Ala Asn Phe Asp Val Leu Asn Asn Ser Ala Thr Asp Leu 550 555 560 AAT AGA CAC GTT GAA GAT GCT TAT AAA GGT TTA TTA AAT CTA AAT GAA 1902 Asn Arg His Val Glu Asp Ala Tyr Lys Gly Leu Leu Asn Leu Asn Glu 565 570 575 580 AAA AAT GCA AAT AAA CAA CCG TTG GTG ACT GAC AGC ACG GCG GCG ACT 1950 Lys Asn Ala Asn Lys Gln Pro Leu Val Thr Asp Ser Thr Ala Ala Thr 585 590 595 GTA GGC GAT TTA CGT AAA TTG GGT TGG GTA GTA TCA ACC AAA AAC GGT 1998 Val Gly Asp Leu Arg Lys Leu Gly Trp Val Val Ser Thr Lys Asn Gly 600 605 610 ACG AAA GAA GAA AGC AAT CAA GTT AAA CAA GCT GAT GAA GTC CTC TTT 2046 Thr Lys Glu Glu Ser Asn Gln Val Lys Gln Ala Asp Glu Val Leu Phe 615 620 625 ACC GGA GCC GGT GCT GCT ACG GTT ACT TCC AAA TCT GAA AAC GGT AAA 2094 Thr Gly Ala Gly Ala Ala Thr Val Thr Ser Lys Ser Glu Asn Gly Lys 630 635 640 CAT ACG ATT ACC GTT AGT GTG GCT GAA ACT AAA GCG GAT TGC GGT CTT 2142 His Thr Ile Thr Val Ser Val Ala Glu Thr Lys Ala Asp Cys Gly Leu 645 650 655 660 GAA AAA GAT GGC GAT ACT ATT AAG CTC AAA GTG GAT AAT CAA AAC ACT 2190 Glu Lys Asp Gly Asp Thr Ile Lys Leu Lys Val Asp Asn Gln Asn Thr 665 670 675 GAT AAT GTT TTA ACT GTT GGT AAT AAT GGT ACT GCT GTC ACT AAA GGT 2238 Asp Asn Val Leu Thr Val Gly Asn Asn Gly Thr Ala Val Thr Lys Gly 680 685 690 GGC TTT GAA ACT GTT AAA ACT GGA GCG ACT GAT GCA GAT CGC GGT AAA 2286 Gly Phe Glu Thr Val Lys Thr Gly Ala Thr Asp Ala Asp Arg Gly Lys 695 700 705 GTA ACT GTA AAA GAT GCT ACT GCT AAT GAC GCT GAT AAG AAA GTC GCA 2334 Val Thr Val Lys Asp Ala Thr Ala Asn Asp Ala Asp Lys Lys Val Ala 710 715 720 ACT GTA AAA GAT GTT GCA ACC GCA ATT AAT AGT GCG GCG ACT TTT GTG 2382 Thr Val Lys Asp Val Ala Thr Ala Ile Asn Ser Ala Ala Thr Phe Val 725 730 735 740 AAA ACA GAG AAT TTA ACT ACC TCT ATT GAT GAA GAT AAT CCT ACA GAT 2430 Lys Thr Glu Asn Leu Thr Thr Ser Ile Asp Glu Asp Asn Pro Thr Asp 745 750 755 AAC GGC AAA GAT GAC GCA CTT AAA GCG GGC GAT ACC TTA ACC TTT AAA 2478 Asn Gly Lys Asp Asp Ala Leu Lys Ala Gly Asp Thr Leu Thr Phe Lys 760 765 770 GCA GGT AAA AAC CTG AAA GTT AAA CGT GAT GGA AAA AAT ATT ACT TTT 2526 Ala Gly Lys Asn Leu Lys Val Lys Arg Asp Gly Lys Asn Ile Thr Phe 775 780 785 GAC TTG GCG AAA AAC CTT GAG GTG AAA ACT GCG AAA GTG AGT GAT ACT 2574 Asp Leu Ala Lys Asn Leu Glu Val Lys Thr Ala Lys Val Ser Asp Thr 790 795 800 TTA ACG ATT GGC GGG AAT ACA CCT ACA GGT GGC ACT ACT GCG ACG CCA 2622 Leu Thr Ile Gly Gly Asn Thr Pro Thr Gly Gly Thr Thr Ala Thr Pro 805 810 815 820 AAA GTG AAT ATT ACT AGC ACG GCT GAT GGT TTG AAT TTT GCA AAA GAA 2670 Lys Val Asn Ile Thr Ser Thr Ala Asp Gly Leu Asn Phe Ala Lys Glu 825 830 835 ACA GCC GAT GCC TCG GGT TCT AAG AAT GTT TAT TTG AAA GGT ATT GCG 2718 Thr Ala Asp Ala Ser Gly Ser Lys Asn Val Tyr Leu Lys Gly Ile Ala 840 845 850 ACA ACT TTA ACT GAG CCA AGC GCG GGA GCG AAG TCT TCA CAC GTT GAT 2766 Thr Thr Leu Thr Glu Pro Ser Ala Gly Ala Lys Ser Ser His Val Asp 855 860 865 TTA AAT GTG GAT GCG ACG AAA AAA TCC AAT GCA GCA AGT ATT GAA GAT 2814 Leu Asn Val Asp Ala Thr Lys Lys Ser Asn Ala Ala Ser Ile Glu Asp 870 875 880 GTA TTG CGC GCA GGT TGG AAT ATT CAA GGT AAT GGT AAT AAT GTT GAT 2862 Val Leu Arg Ala Gly Trp Asn Ile Gln Gly Asn Gly Asn Asn Val Asp 885 890 895 900 TAT GTA GCG ACG TAT GAC ACA GTA AAC TTT ACC GAT GAC AGC ACA GGT 2910 Tyr Val Ala Thr Tyr Asp Thr Val Asn Phe Thr Asp Asp Ser Thr Gly 905 910 915 ACA ACA ACG GTA ACC GTA ACC CAA AAA GCA GAT GGC AAA GGT GCT GAC 2958 Thr Thr Thr Val Thr Val Thr Gln Lys Ala Asp Gly Lys Gly Ala Asp 920 925 930 GTT AAA ATC GGT GCG AAA ACT TCT GTT ATC AAA GAC CAC AAC GGC AAA 3006 Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Asp His Asn Gly Lys 935 940 945 CTG TTT ACA GGC AAA GAC CTG AAA GAT GCG AAT AAT GGT GCA ACC GTT 3054 Leu Phe Thr Gly Lys Asp Leu Lys Asp Ala Asn Asn Gly Ala Thr Val 950 955 960 AGT GAA GAT GAT GGC AAA GAC ACC GGC ACA GGC TTA GTT ACT GCA AAA 3102 Ser Glu Asp Asp Gly Lys Asp Thr Gly Thr Gly Leu Val Thr Ala Lys 965 970 975 980 ACT GTG ATT GAT GCA GTA AAT AAA AGC GGT TGG AGG GTA ACC GGT GAG 3150 Thr Val Ile Asp Ala Val Asn Lys Ser Gly Trp Arg Val Thr Gly Glu 985 990 995 GGC GCG ACT GCC GAA ACC GGT GCA ACC GCC GTG AAT GCG GGT AAC GCT 3198 Gly Ala Thr Ala Glu Thr Gly Ala Thr Ala Val Asn Ala Gly Asn Ala 1000 1005 1010 GAA ACC GTT ACA TCA GGC ACG AGC GTG AAC TTC AAA AAC GGC AAT GCG 3246 Glu Thr Val Thr Ser Gly Thr Ser Val Asn Phe Lys Asn Gly Asn Ala 1015 1020 1025 ACC ACA GCG ACC GTA AGC AAA GAT AAT GGC AAC ATC AAT GTC AAA TAC 3294 Thr Thr Ala Thr Val Ser Lys Asp Asn Gly Asn Ile Asn Val Lys Tyr 1030 1035 1040 GAT GTA AAT GTT GGT GAC GGC TTG AAG ATT GGC GAT GAC AAA AAA ATC 3342 Asp Val Asn Val Gly Asp Gly Leu Lys Ile Gly Asp Asp Lys Lys Ile 1045 1050 1055 1060 GTT GCA GAC ACG ACC ACA CTT ACT GTA ACA GGT GGT AAG GTG TCT GTT 3390 Val Ala Asp Thr Thr Thr Leu Thr Val Thr Gly Gly Lys Val Ser Val 1065 1070 1075 CCT GCT GGT GCT AAT AGT GTT AAT AAC AAT AAG AAA CTT GTT AAT GCA 3438 Pro Ala Gly Ala Asn Ser Val Asn Asn Asn Lys Lys Leu Val Asn Ala 1080 1085 1090 GAG GGT TTA GCG ACT GCT TTA AAC AAC CTA AGC TGG ACG GCA AAA GCC 3486 Glu Gly Leu Ala Thr Ala Leu Asn Asn Leu Ser Trp Thr Ala Lys Ala 1095 1100 1105 GAT AAA TAT GCA GAT GGC GAG TCA GAG GGC GAA ACC GAC CAA GAA GTC 3534 Asp Lys Tyr Ala Asp Gly Glu Ser Glu Gly Glu Thr Asp Gln Glu Val 1110 1115 1120 AAA GCA GGC GAC AAA GTA ACC TTT AAA GCA GGC AAG AAC TTA AAA GTG 3582 Lys Ala Gly Asp Lys Val Thr Phe Lys Ala Gly Lys Asn Leu Lys Val 1125 1130 1135 1140 AAA CAG TCT GAA AAA GAC TTT ACT TAT TCA CTG CAA GAC ACT TTA ACA 3630 Lys Gln Ser Glu Lys Asp Phe Thr Tyr Ser Leu Gln Asp Thr Leu Thr 1145 1150 1155 GGC TTA ACG AGC ATT ACT TTA GGT GGT ACA GCT AAT GGC AGA AAT GAT 3678 Gly Leu Thr Ser Ile Thr Leu Gly Gly Thr Ala Asn Gly Arg Asn Asp 1160 1165 1170 ACG GGA ACC GTC ATC AAC AAA GAC GGC TTA ACC ATC ACG CTG GCA AAT 3726 Thr Gly Thr Val Ile Asn Lys Asp Gly Leu Thr Ile Thr Leu Ala Asn 1175 1180 1185 GGT GCT GCG GCA GGC ACA GAT GCG TCT AAC GGA AAC ACC ATC AGT GTA 3774 Gly Ala Ala Ala Gly Thr Asp Ala Ser Asn Gly Asn Thr Ile Ser Val 1190 1195 1200 ACC AAA GAC GGC ATT AGT GCG GGT AAT AAA GAA ATT ACC AAT GTT AAG 3822 Thr Lys Asp Gly Ile Ser Ala Gly Asn Lys Glu Ile Thr Asn Val Lys 1205 1210 1215 1220 AGT GCT TTA AAA ACC TAT AAA GAT ACT CAA AAC ACT GCA GAT GAA ACA 3870 Ser Ala Leu Lys Thr Tyr Lys Asp Thr Gln Asn Thr Ala Asp Glu Thr 1225 1230 1235 CAA GAT AAA GAG TTC CAC GCC GCC GTT AAA AAC GCA AAT GAA GTT GAG 3918 Gln Asp Lys Glu Phe His Ala Ala Val Lys Asn Ala Asn Glu Val Glu 1240 1245 1250 TTC GTG GGT AAA AAC GGT GCA ACC GTG TCT GCA AAA ACT GAT AAC AAC 3966 Phe Val Gly Lys Asn Gly Ala Thr Val Ser Ala Lys Thr Asp Asn Asn 1255 1260 1265 GGA AAA CAT ACT GTA ACG ATT GAT GTT GCA GAA GCC AAA GTT GGT GAT 4014 Gly Lys His Thr Val Thr Ile Asp Val Ala Glu Ala Lys Val Gly Asp 1270 1275 1280 GGT CTT GAA AAA GAT ACT GAC GGC AAG ATT AAA CTC AAA GTA GAT AAT 4062 Gly Leu Glu Lys Asp Thr Asp Gly Lys Ile Lys Leu Lys Val Asp Asn 1285 1290 1295 1300 ACA GAT GGG AAT AAT CTA TTA ACC GTT GAT GCA ACA AAA GGT GCA TCC 4110 Thr Asp Gly Asn Asn Leu Leu Thr Val Asp Ala Thr Lys Gly Ala Ser 1305 1310 1315 GTT GCC AAG GGC GAG TTT AAT GCC GTA ACA ACA GAT GCA ACT ACA GCC 4158 Val Ala Lys Gly Glu Phe Asn Ala Val Thr Thr Asp Ala Thr Thr Ala 1320 1325 1330 CAA GGC ACA AAT GCC AAT GAG CGC GGT AAA GTG GTT GTC AAG GGT TCA 4206 Gln Gly Thr Asn Ala Asn Glu Arg Gly Lys Val Val Val Lys Gly Ser 1335 1340 1345 AAT GGT GCA ACT GCT ACC GAA ACT GAC AAG AAA AAA GTG GCA ACT GTT 4254 Asn Gly Ala Thr Ala Thr Glu Thr Asp Lys Lys Lys Val Ala Thr Val 1350 1355 1360 GGC GAC GTT GCT AAA GCG ATT AAC GAC GCA GCA ACT TTC GTG AAA GTG 4302 Gly Asp Val Ala Lys Ala Ile Asn Asp Ala Ala Thr Phe Val Lys Val 1365 1370 1375 1380 GAA AAT GAC GAC AGT GCT ACG ATT GAT GAT AGC CCA ACA GAT GAT GGC 4350 Glu Asn Asp Asp Ser Ala Thr Ile Asp Asp Ser Pro Thr Asp Asp Gly 1385 1390 1395 GCA AAT GAT GCT CTC AAA GCA GGC GAC ACC TTG ACC TTA AAA GCG GGT 4398 Ala Asn Asp Ala Leu Lys Ala Gly Asp Thr Leu Thr Leu Lys Ala Gly 1400 1405 1410 AAA AAC TTA AAA GTT AAA CGT GAT GGT AAA AAT ATT ACT TTT GCC CTT 4446 Lys Asn Leu Lys Val Lys Arg Asp Gly Lys Asn Ile Thr Phe Ala Leu 1415 1420 1425 GCG AAC GAC CTT AGT GTA AAA AGC GCA ACC GTT AGC GAT AAA TTA TCG 4494 Ala Asn Asp Leu Ser Val Lys Ser Ala Thr Val Ser Asp Lys Leu Ser 1430 1435 1440 CTT GGT ACA AAC GGC AAT AAA GTC AAT ATC ACA AGC GAC ACC AAA GGC 4542 Leu Gly Thr Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr Lys Gly 1445 1450 1455 1460 TTG AAC TTC GCT AAA GAT AGT AAG ACA GGC GAT GAT GCT AAT ATT CAC 4590 Leu Asn Phe Ala Lys Asp Ser Lys Thr Gly Asp Asp Ala Asn Ile His 1465 1470 1475 TTA AAT GGC ATT GCT TCA ACT TTA ACT GAT ACA TTG TTA AAT AGT GGT 4638 Leu Asn Gly Ile Ala Ser Thr Leu Thr Asp Thr Leu Leu Asn Ser Gly 1480 1485 1490 GCG ACA ACC AAT TTA GGT GGT AAT GGT ATT ACT GAT AAC GAG AAA AAA 4686 Ala Thr Thr Asn Leu Gly Gly Asn Gly Ile Thr Asp Asn Glu Lys Lys 1495 1500 1505 CGC GCG GCG AGC GTT AAA GAT GTC TTG AAT GCG GGT TGG AAT GTT CGT 4734 Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn Val Arg 1510 1515 1520 GGT GTT AAA CCG GCA TCT GCA AAT AAT CAA GTG GAG AAT ATC GAC TTT 4782 Gly Val Lys Pro Ala Ser Ala Asn Asn Gln Val Glu Asn Ile Asp Phe 1525 1530 1535 1540 GTA GCA ACC TAC GAC ACA GTG GAC TTT GTT AGT GGA GAT AAA GAC ACC 4830 Val Ala Thr Tyr Asp Thr Val Asp Phe Val Ser Gly Asp Lys Asp Thr 1545 1550 1555 ACG AGT GTA ACT GTT GAA AGT AAA GAT AAT GGC AAG AGA ACC GAA GTT 4878 Thr Ser Val Thr Val Glu Ser Lys Asp Asn Gly Lys Arg Thr Glu Val 1560 1565 1570 AAA ATC GGT GCG AAG ACT TCT GTT ATC AAA GAC CAC AAC GGC AAA CTG 4926 Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Asp His Asn Gly Lys Leu 1575 1580 1585 TTT ACA GGC AAA GAG CTG AAG GAT GCT AAC AAT AAT GGC GTA ACT GTT 4974 Phe Thr Gly Lys Glu Leu Lys Asp Ala Asn Asn Asn Gly Val Thr Val 1590 1595 1600 ACC GAA ACC GAC GGC AAA GAC GAG GGT AAT GGT TTA GTG ACT GCA AAA 5022 Thr Glu Thr Asp Gly Lys Asp Glu Gly Asn Gly Leu Val Thr Ala Lys 1605 1610 1615 1620 GCT GTG ATT GAT GCC GTG AAT AAG GCT GGT TGG AGA GTT AAA ACA ACA 5070 Ala Val Ile Asp Ala Val Asn Lys Ala Gly Trp Arg Val Lys Thr Thr 1625 1630 1635 GGT GCT AAT GGT CAG AAT GAT GAC TTC GCA ACT GTT GCG TCA GGC ACA 5118 Gly Ala Asn Gly Gln Asn Asp Asp Phe Ala Thr Val Ala Ser Gly Thr 1640 1645 1650 AAT GTA ACC TTT GCT GAT GGT AAT GGC ACA ACT GCC GAA GTA ACT AAA 5166 Asn Val Thr Phe Ala Asp Gly Asn Gly Thr Thr Ala Glu Val Thr Lys 1655 1660 1665 GCA AAC GAC GGT AGT ATT ACT GTT AAA TAC AAT GTT AAA GTG GCT GAT 5214 Ala Asn Asp Gly Ser Ile Thr Val Lys Tyr Asn Val Lys Val Ala Asp 1670 1675 1680 GGC TTA AAA CTA GAC GGC GAT AAA ATC GTT GCA GAC ACG ACC GTA CTT 5262 Gly Leu Lys Leu Asp Gly Asp Lys Ile Val Ala Asp Thr Thr Val Leu 1685 1690 1695 1700 ACT GTG GCA GAT GGT AAA GTT ACA GCT CCG AAT AAT GGC GAT GGT AAG 5310 Thr Val Ala Asp Gly Lys Val Thr Ala Pro Asn Asn Gly Asp Gly Lys 1705 1710 1715 AAA TTT GTT GAT GCA AGT GGT TTA GCG GAT GCG TTA AAT AAA TTA AGC 5358 Lys Phe Val Asp Ala Ser Gly Leu Ala Asp Ala Leu Asn Lys Leu Ser 1720 1725 1730 TGG ACG GCA ACT GCT GGT AAA GAA GGC ACT GGT GAA GTT GAT CCT GCA 5406 Trp Thr Ala Thr Ala Gly Lys Glu Gly Thr Gly Glu Val Asp Pro Ala 1735 1740 1745 AAT TCA GCA GGG CAA GAA GTC AAA GCG GGC GAC AAA GTA ACC TTT AAA 5454 Asn Ser Ala Gly Gln Glu Val Lys Ala Gly Asp Lys Val Thr Phe Lys 1750 1755 1760 GCC GGC GAC AAC CTG AAA ATC AAA CAA AGC GGC AAA GAC TTT ACC TAC 5502 Ala Gly Asp Asn Leu Lys Ile Lys Gln Ser Gly Lys Asp Phe Thr Tyr 1765 1770 1775 1780 TCG CTG AAA AAA GAG CTG AAA GAC CTG ACC AGC GTA GAG TTC AAA GAC 5550 Ser Leu Lys Lys Glu Leu Lys Asp Leu Thr Ser Val Glu Phe Lys Asp 1785 1790 1795 GCA AAC GGC GGT ACA GGC AGT GAA AGC ACC AAG ATT ACC AAA GAC GGC 5598 Ala Asn Gly Gly Thr Gly Ser Glu Ser Thr Lys Ile Thr Lys Asp Gly 1800 1805 1810 TTG ACC ATT ACG CCG GCA AAC GGT GCG GGT GCG GCA GGT GCA AAC ACT 5646 Leu Thr Ile Thr Pro Ala Asn Gly Ala Gly Ala Ala Gly Ala Asn Thr 1815 1820 1825 GCA AAC ACC ATT AGC GTA ACC AAA GAT GGC ATT AGC GCG GGT AAT AAA 5694 Ala Asn Thr Ile Ser Val Thr Lys Asp Gly Ile Ser Ala Gly Asn Lys 1830 1835 1840 GCA GTT ACA AAC GTT GTG AGC GGA CTG AAG AAA TTT GGT GAT GGT CAT 5742 Ala Val Thr Asn Val Val Ser Gly Leu Lys Lys Phe Gly Asp Gly His 1845 1850 1855 1860 ACG TTG GCA AAT GGC ACT GTT GCT GAT TTT GAA AAG CAT TAT GAC AAT 5790 Thr Leu Ala Asn Gly Thr Val Ala Asp Phe Glu Lys His Tyr Asp Asn 1865 1870 1875 GCC TAT AAA GAC TTG ACC AAT TTG GAT GAA AAA GGC GCG GAT AAT AAT 5838 Ala Tyr Lys Asp Leu Thr Asn Leu Asp Glu Lys Gly Ala Asp Asn Asn 1880 1885 1890 CCG ACT GTT GCC GAC AAT ACC GCT GCA ACC GTG GGC GAT TTG CGC GGC 5886 Pro Thr Val Ala Asp Asn Thr Ala Ala Thr Val Gly Asp Leu Arg Gly 1895 1900 1905 TTG GGC TGG GTC ATT TCT GCG GAC AAA ACC ACA GGC GAA CCC AAT CAG 5934 Leu Gly Trp Val Ile Ser Ala Asp Lys Thr Thr Gly Glu Pro Asn Gln 1910 1915 1920 GAA TAC AAC GCG CAA GTG CGT AAC GCC AAT GAA GTG AAA TTC AAG AGC 5982 Glu Tyr Asn Ala Gln Val Arg Asn Ala Asn Glu Val Lys Phe Lys Ser 1925 1930 1935 1940 GGC AAC GGT ATC AAT GTT TCC GGT AAA ACA TTG AAC GGT ACG CGC GTG 6030 Gly Asn Gly Ile Asn Val Ser Gly Lys Thr Leu Asn Gly Thr Arg Val 1945 1950 1955 ATT ACC TTT GAA TTG GCT AAA GGC GAA GTG GTT AAA TCG AAT GAA TTT 6078 Ile Thr Phe Glu Leu Ala Lys Gly Glu Val Val Lys Ser Asn Glu Phe 1960 1965 1970 ACC GTT AAG AAT GCC GAT GGT TCG GAA ACG AAC TTG GTT AAA GTT GGC 6126 Thr Val Lys Asn Ala Asp Gly Ser Glu Thr Asn Leu Val Lys Val Gly 1975 1980 1985 GAT ATG TAT TAC AGC AAA GAG GAT ATT GAC CCG GCA ACC AGT AAA CCG 6174 Asp Met Tyr Tyr Ser Lys Glu Asp Ile Asp Pro Ala Thr Ser Lys Pro 1990 1995 2000 ATG ACA GGT AAA ACT GAA AAA TAT AAG GTT GAA AAC GGC AAA GTC GTT 6222 Met Thr Gly Lys Thr Glu Lys Tyr Lys Val Glu Asn Gly Lys Val Val 2005 2010 2015 2020 TCT GCT AAC GGC AGC AAG ACC GAA GTT ACC CTA ACC AAC AAA GGT TCC 6270 Ser Ala Asn Gly Ser Lys Thr Glu Val Thr Leu Thr Asn Lys Gly Ser 2025 2030 2035 GGC TAT GTA ACA GGT AAC CAA GTG GCT GAT GCG ATT GCG AAA TCA GGC 6318 Gly Tyr Val Thr Gly Asn Gln Val Ala Asp Ala Ile Ala Lys Ser Gly 2040 2045 2050 TTT GAG CTT GGT TTG GCT GAT GCG GCA GAA GCT GAA AAA GCC TTT GCA 6366 Phe Glu Leu Gly Leu Ala Asp Ala Ala Glu Ala Glu Lys Ala Phe Ala 2055 2060 2065 GAA AGC GCA AAA GAC AAG CAA TTG TCT AAA GAT AAA GCG GAA ACT GTA 6414 Glu Ser Ala Lys Asp Lys Gln Leu Ser Lys Asp Lys Ala Glu Thr Val 2070 2075 2080 AAT GCC CAC GAT AAA GTC CGT TTT GCT AAT GGT TTA AAT ACC AAA GTG 6462 Asn Ala His Asp Lys Val Arg Phe Ala Asn Gly Leu Asn Thr Lys Val 2085 2090 2095 2100 AGC GCG GCA ACG GTG GAA AGC ACT GAT GCA AAC GGC GAT AAA GTG ACC 6510 Ser Ala Ala Thr Val Glu Ser Thr Asp Ala Asn Gly Asp Lys Val Thr 2105 2110 2115 ACA ACC TTT GTG AAA ACC GAT GTG GAA TTG CCT TTA ACG CAA ATC TAC 6558 Thr Thr Phe Val Lys Thr Asp Val Glu Leu Pro Leu Thr Gln Ile Tyr 2120 2125 2130 AAT ACC GAT GCA AAC GGT AAT AAG ATC GTT AAA AAA GCT GAC GGA AAA 6606 Asn Thr Asp Ala Asn Gly Asn Lys Ile Val Lys Lys Ala Asp Gly Lys 2135 2140 2145 TGG TAT GAA CTG AAT GCT GAT GGT ACG GCG AGT AAC AAA GAA GTG ACA 6654 Trp Tyr Glu Leu Asn Ala Asp Gly Thr Ala Ser Asn Lys Glu Val Thr 2150 2155 2160 CTT GGT AAC GTG GAT GCA AAC GGT AAG AAA GTT GTG AAA GTA ACC GAA 6702 Leu Gly Asn Val Asp Ala Asn Gly Lys Lys Val Val Lys Val Thr Glu 2165 2170 2175 2180 AAT GGT GCG GAT AAG TGG TAT TAC ACC AAT GCT GAC GGT GCT GCG GAT 6750 Asn Gly Ala Asp Lys Trp Tyr Tyr Thr Asn Ala Asp Gly Ala Ala Asp 2185 2190 2195 AAA ACC AAA GGC GAA GTG AGC AAT GAT AAA GTT TCT ACC GAT GAA AAA 6798 Lys Thr Lys Gly Glu Val Ser Asn Asp Lys Val Ser Thr Asp Glu Lys 2200 2205 2210 CAC GTT GTC CGC CTT GAT CCG AAC AAT CAA TCG AAC GGC AAA GGC GTG 6846 His Val Val Arg Leu Asp Pro Asn Asn Gln Ser Asn Gly Lys Gly Val 2215 2220 2225 GTC ATT GAC AAT GTG GCT AAT GGC GAA ATT TCT GCC ACT TCC ACC GAT 6894 Val Ile Asp Asn Val Ala Asn Gly Glu Ile Ser Ala Thr Ser Thr Asp 2230 2235 2240 GCG ATT AAC GGA AGT CAG TTG TAT GCC GTG GCA AAA GGG GTA ACA AAC 6942 Ala Ile Asn Gly Ser Gln Leu Tyr Ala Val Ala Lys Gly Val Thr Asn 2245 2250 2255 2260 CTT GCT GGA CAA GTG AAT AAT CTT GAG GGC AAA GTG AAT AAA GTG GGC 6990 Leu Ala Gly Gln Val Asn Asn Leu Glu Gly Lys Val Asn Lys Val Gly 2265 2270 2275 AAA CGT GCA GAT GCA GGT ACA GCA AGT GCA TTA GCG GCT TCA CAG TTA 7038 Lys Arg Ala Asp Ala Gly Thr Ala Ser Ala Leu Ala Ala Ser Gln Leu 2280 2285 2290 CCA CAA GCC ACT ATG CCA GGT AAA TCA ATG GTT GCT ATT GCG GGA AGT 7086 Pro Gln Ala Thr Met Pro Gly Lys Ser Met Val Ala Ile Ala Gly Ser 2295 2300 2305 AGT TAT CAA GGT CAA AAT GGT TTA GCT ATC GGG GTA TCA AGA ATT TCC 7134 Ser Tyr Gln Gly Gln Asn Gly Leu Ala Ile Gly Val Ser Arg Ile Ser 2310 2315 2320 GAT AAT GGC AAA GTG ATT ATT CGC TTG TCA GGC ACA ACC AAT AGT CAA 7182 Asp Asn Gly Lys Val Ile Ile Arg Leu Ser Gly Thr Thr Asn Ser Gln 2325 2330 2335 2340 GGT AAA ACA GGC GTT GCA GCA GGT GTT GGT TAC CAG TGG TAAAGTTTGG 7231 Gly Lys Thr Gly Val Ala Ala Gly Val Gly Tyr Gln Trp 2345 2350 ATTATCTCTC TTAAAAAGCG GCATTTGCCG CTTTTTTTAT GGGTGGCTAT TATGTATCGT 7291 2353 amino acids amino acid linear protein unknown 4 Met Asn Lys Ile Phe Asn Val Ile Trp Asn Val Met Thr Gln Thr Trp 1 5 10 15 Val Val Val Ser Glu Leu Thr Arg Thr His Thr Lys Arg Ala Ser Ala 20 25 30 Thr Val Glu Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45 Ala Asn Ala Thr Asp Glu Asp Glu Glu Leu Asp Pro Val Val Arg Thr 50 55 60 Ala Pro Val Leu Ser Phe His Ser Asp Lys Glu Gly Thr Gly Glu Lys 65 70 75 80 Glu Val Thr Glu Asn Ser Asn Trp Gly Ile Tyr Phe Asp Asn Lys Gly 85 90 95 Val Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu Lys 100 105 110 Ile Lys Gln Asn Thr Asp Glu Ser Thr Asn Ala Ser Ser Phe Thr Tyr 115 120 125 Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Ala Thr Glu Lys 130 135 140 Leu Ser Phe Gly Ala Asn Gly Asp Lys Val Asp Ile Thr Ser Asp Ala 145 150 155 160 Asn Gly Leu Lys Leu Ala Lys Thr Gly Asn Gly Asn Val His Leu Asn 165 170 175 Gly Leu Asp Ser Thr Leu Pro Asp Ala Val Thr Asn Thr Gly Val Leu 180 185 190 Ser Ser Ser Ser Phe Thr Pro Asn Asp Val Glu Lys Thr Arg Ala Ala 195 200 205 Thr Val Lys Asp Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Ala Lys 210 215 220 Thr Ala Gly Gly Asn Val Glu Ser Val Asp Leu Val Ser Ala Tyr Asn 225 230 235 240 Asn Val Glu Phe Ile Thr Gly Asp Lys Asn Thr Leu Asp Val Val Leu 245 250 255 Thr Ala Lys Glu Asn Gly Lys Thr Thr Glu Val Lys Phe Thr Pro Lys 260 265 270 Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu Phe Thr Gly Lys Glu 275 280 285 Asn Asn Asp Thr Asn Lys Val Thr Ser Asn Thr Ala Thr Asp Asn Thr 290 295 300 Asp Glu Gly Asn Gly Leu Val Thr Ala Lys Ala Val Ile Asp Ala Val 305 310 315 320 Asn Lys Ala Gly Trp Arg Val Lys Thr Thr Thr Ala Asn Gly Gln Asn 325 330 335 Gly Asp Phe Ala Thr Val Ala Ser Gly Thr Asn Val Thr Phe Glu Ser 340 345 350 Gly Asp Gly Thr Thr Ala Ser Val Thr Lys Asp Thr Asn Gly Asn Gly 355 360 365 Ile Thr Val Lys Tyr Asp Ala Lys Val Gly Asp Gly Leu Lys Phe Asp 370 375 380 Ser Asp Lys Lys Ile Val Ala Asp Thr Thr Ala Leu Thr Val Thr Gly 385 390 395 400 Gly Lys Val Ala Glu Ile Ala Lys Glu Asp Asp Lys Lys Lys Leu Val 405 410 415 Asn Ala Gly Asp Leu Val Thr Ala Leu Gly Asn Leu Ser Trp Lys Ala 420 425 430 Lys Ala Glu Ala Asp Thr Asp Gly Ala Leu Glu Gly Ile Ser Lys Asp 435 440 445 Gln Glu Val Lys Ala Gly Glu Thr Val Thr Phe Lys Ala Gly Lys Asn 450 455 460 Leu Lys Val Lys Gln Asp Gly Ala Asn Phe Thr Tyr Ser Leu Gln Asp 465 470 475 480 Ala Leu Thr Gly Leu Thr Ser Ile Thr Leu Gly Gly Thr Thr Asn Gly 485 490 495 Gly Asn Asp Ala Lys Thr Val Ile Asn Lys Asp Gly Leu Thr Ile Thr 500 505 510 Pro Ala Gly Asn Gly Gly Thr Thr Gly Thr Asn Thr Ile Ser Val Thr 515 520 525 Lys Asp Gly Ile Lys Ala Gly Asn Lys Ala Ile Thr Asn Val Ala Ser 530 535 540 Gly Leu Arg Ala Tyr Asp Asp Ala Asn Phe Asp Val Leu Asn Asn Ser 545 550 555 560 Ala Thr Asp Leu Asn Arg His Val Glu Asp Ala Tyr Lys Gly Leu Leu 565 570 575 Asn Leu Asn Glu Lys Asn Ala Asn Lys Gln Pro Leu Val Thr Asp Ser 580 585 590 Thr Ala Ala Thr Val Gly Asp Leu Arg Lys Leu Gly Trp Val Val Ser 595 600 605 Thr Lys Asn Gly Thr Lys Glu Glu Ser Asn Gln Val Lys Gln Ala Asp 610 615 620 Glu Val Leu Phe Thr Gly Ala Gly Ala Ala Thr Val Thr Ser Lys Ser 625 630 635 640 Glu Asn Gly Lys His Thr Ile Thr Val Ser Val Ala Glu Thr Lys Ala 645 650 655 Asp Cys Gly Leu Glu Lys Asp Gly Asp Thr Ile Lys Leu Lys Val Asp 660 665 670 Asn Gln Asn Thr Asp Asn Val Leu Thr Val Gly Asn Asn Gly Thr Ala 675 680 685 Val Thr Lys Gly Gly Phe Glu Thr Val Lys Thr Gly Ala Thr Asp Ala 690 695 700 Asp Arg Gly Lys Val Thr Val Lys Asp Ala Thr Ala Asn Asp Ala Asp 705 710 715 720 Lys Lys Val Ala Thr Val Lys Asp Val Ala Thr Ala Ile Asn Ser Ala 725 730 735 Ala Thr Phe Val Lys Thr Glu Asn Leu Thr Thr Ser Ile Asp Glu Asp 740 745 750 Asn Pro Thr Asp Asn Gly Lys Asp Asp Ala Leu Lys Ala Gly Asp Thr 755 760 765 Leu Thr Phe Lys Ala Gly Lys Asn Leu Lys Val Lys Arg Asp Gly Lys 770 775 780 Asn Ile Thr Phe Asp Leu Ala Lys Asn Leu Glu Val Lys Thr Ala Lys 785 790 795 800 Val Ser Asp Thr Leu Thr Ile Gly Gly Asn Thr Pro Thr Gly Gly Thr 805 810 815 Thr Ala Thr Pro Lys Val Asn Ile Thr Ser Thr Ala Asp Gly Leu Asn 820 825 830 Phe Ala Lys Glu Thr Ala Asp Ala Ser Gly Ser Lys Asn Val Tyr Leu 835 840 845 Lys Gly Ile Ala Thr Thr Leu Thr Glu Pro Ser Ala Gly Ala Lys Ser 850 855 860 Ser His Val Asp Leu Asn Val Asp Ala Thr Lys Lys Ser Asn Ala Ala 865 870 875 880 Ser Ile Glu Asp Val Leu Arg Ala Gly Trp Asn Ile Gln Gly Asn Gly 885 890 895 Asn Asn Val Asp Tyr Val Ala Thr Tyr Asp Thr Val Asn Phe Thr Asp 900 905 910 Asp Ser Thr Gly Thr Thr Thr Val Thr Val Thr Gln Lys Ala Asp Gly 915 920 925 Lys Gly Ala Asp Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Asp 930 935 940 His Asn Gly Lys Leu Phe Thr Gly Lys Asp Leu Lys Asp Ala Asn Asn 945 950 955 960 Gly Ala Thr Val Ser Glu Asp Asp Gly Lys Asp Thr Gly Thr Gly Leu 965 970 975 Val Thr Ala Lys Thr Val Ile Asp Ala Val Asn Lys Ser Gly Trp Arg 980 985 990 Val Thr Gly Glu Gly Ala Thr Ala Glu Thr Gly Ala Thr Ala Val Asn 995 1000 1005 Ala Gly Asn Ala Glu Thr Val Thr Ser Gly Thr Ser Val Asn Phe Lys 1010 1015 1020 Asn Gly Asn Ala Thr Thr Ala Thr Val Ser Lys Asp Asn Gly Asn Ile 1025 1030 1035 1040 Asn Val Lys Tyr Asp Val Asn Val Gly Asp Gly Leu Lys Ile Gly Asp 1045 1050 1055 Asp Lys Lys Ile Val Ala Asp Thr Thr Thr Leu Thr Val Thr Gly Gly 1060 1065 1070 Lys Val Ser Val Pro Ala Gly Ala Asn Ser Val Asn Asn Asn Lys Lys 1075 1080 1085 Leu Val Asn Ala Glu Gly Leu Ala Thr Ala Leu Asn Asn Leu Ser Trp 1090 1095 1100 Thr Ala Lys Ala Asp Lys Tyr Ala Asp Gly Glu Ser Glu Gly Glu Thr 1105 1110 1115 1120 Asp Gln Glu Val Lys Ala Gly Asp Lys Val Thr Phe Lys Ala Gly Lys 1125 1130 1135 Asn Leu Lys Val Lys Gln Ser Glu Lys Asp Phe Thr Tyr Ser Leu Gln 1140 1145 1150 Asp Thr Leu Thr Gly Leu Thr Ser Ile Thr Leu Gly Gly Thr Ala Asn 1155 1160 1165 Gly Arg Asn Asp Thr Gly Thr Val Ile Asn Lys Asp Gly Leu Thr Ile 1170 1175 1180 Thr Leu Ala Asn Gly Ala Ala Ala Gly Thr Asp Ala Ser Asn Gly Asn 1185 1190 1195 1200 Thr Ile Ser Val Thr Lys Asp Gly Ile Ser Ala Gly Asn Lys Glu Ile 1205 1210 1215 Thr Asn Val Lys Ser Ala Leu Lys Thr Tyr Lys Asp Thr Gln Asn Thr 1220 1225 1230 Ala Asp Glu Thr Gln Asp Lys Glu Phe His Ala Ala Val Lys Asn Ala 1235 1240 1245 Asn Glu Val Glu Phe Val Gly Lys Asn Gly Ala Thr Val Ser Ala Lys 1250 1255 1260 Thr Asp Asn Asn Gly Lys His Thr Val Thr Ile Asp Val Ala Glu Ala 1265 1270 1275 1280 Lys Val Gly Asp Gly Leu Glu Lys Asp Thr Asp Gly Lys Ile Lys Leu 1285 1290 1295 Lys Val Asp Asn Thr Asp Gly Asn Asn Leu Leu Thr Val Asp Ala Thr 1300 1305 1310 Lys Gly Ala Ser Val Ala Lys Gly Glu Phe Asn Ala Val Thr Thr Asp 1315 1320 1325 Ala Thr Thr Ala Gln Gly Thr Asn Ala Asn Glu Arg Gly Lys Val Val 1330 1335 1340 Val Lys Gly Ser Asn Gly Ala Thr Ala Thr Glu Thr Asp Lys Lys Lys 1345 1350 1355 1360 Val Ala Thr Val Gly Asp Val Ala Lys Ala Ile Asn Asp Ala Ala Thr 1365 1370 1375 Phe Val Lys Val Glu Asn Asp Asp Ser Ala Thr Ile Asp Asp Ser Pro 1380 1385 1390 Thr Asp Asp Gly Ala Asn Asp Ala Leu Lys Ala Gly Asp Thr Leu Thr 1395 1400 1405 Leu Lys Ala Gly Lys Asn Leu Lys Val Lys Arg Asp Gly Lys Asn Ile 1410 1415 1420 Thr Phe Ala Leu Ala Asn Asp Leu Ser Val Lys Ser Ala Thr Val Ser 1425 1430 1435 1440 Asp Lys Leu Ser Leu Gly Thr Asn Gly Asn Lys Val Asn Ile Thr Ser 1445 1450 1455 Asp Thr Lys Gly Leu Asn Phe Ala Lys Asp Ser Lys Thr Gly Asp Asp 1460 1465 1470 Ala Asn Ile His Leu Asn Gly Ile Ala Ser Thr Leu Thr Asp Thr Leu 1475 1480 1485 Leu Asn Ser Gly Ala Thr Thr Asn Leu Gly Gly Asn Gly Ile Thr Asp 1490 1495 1500 Asn Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly 1505 1510 1515 1520 Trp Asn Val Arg Gly Val Lys Pro Ala Ser Ala Asn Asn Gln Val Glu 1525 1530 1535 Asn Ile Asp Phe Val Ala Thr Tyr Asp Thr Val Asp Phe Val Ser Gly 1540 1545 1550 Asp Lys Asp Thr Thr Ser Val Thr Val Glu Ser Lys Asp Asn Gly Lys 1555 1560 1565 Arg Thr Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Asp His 1570 1575 1580 Asn Gly Lys Leu Phe Thr Gly Lys Glu Leu Lys Asp Ala Asn Asn Asn 1585 1590 1595 1600 Gly Val Thr Val Thr Glu Thr Asp Gly Lys Asp Glu Gly Asn Gly Leu 1605 1610 1615 Val Thr Ala Lys Ala Val Ile Asp Ala Val Asn Lys Ala Gly Trp Arg 1620 1625 1630 Val Lys Thr Thr Gly Ala Asn Gly Gln Asn Asp Asp Phe Ala Thr Val 1635 1640 1645 Ala Ser Gly Thr Asn Val Thr Phe Ala Asp Gly Asn Gly Thr Thr Ala 1650 1655 1660 Glu Val Thr Lys Ala Asn Asp Gly Ser Ile Thr Val Lys Tyr Asn Val 1665 1670 1675 1680 Lys Val Ala Asp Gly Leu Lys Leu Asp Gly Asp Lys Ile Val Ala Asp 1685 1690 1695 Thr Thr Val Leu Thr Val Ala Asp Gly Lys Val Thr Ala Pro Asn Asn 1700 1705 1710 Gly Asp Gly Lys Lys Phe Val Asp Ala Ser Gly Leu Ala Asp Ala Leu 1715 1720 1725 Asn Lys Leu Ser Trp Thr Ala Thr Ala Gly Lys Glu Gly Thr Gly Glu 1730 1735 1740 Val Asp Pro Ala Asn Ser Ala Gly Gln Glu Val Lys Ala Gly Asp Lys 1745 1750 1755 1760 Val Thr Phe Lys Ala Gly Asp Asn Leu Lys Ile Lys Gln Ser Gly Lys 1765 1770 1775 Asp Phe Thr Tyr Ser Leu Lys Lys Glu Leu Lys Asp Leu Thr Ser Val 1780 1785 1790 Glu Phe Lys Asp Ala Asn Gly Gly Thr Gly Ser Glu Ser Thr Lys Ile 1795 1800 1805 Thr Lys Asp Gly Leu Thr Ile Thr Pro Ala Asn Gly Ala Gly Ala Ala 1810 1815 1820 Gly Ala Asn Thr Ala Asn Thr Ile Ser Val Thr Lys Asp Gly Ile Ser 1825 1830 1835 1840 Ala Gly Asn Lys Ala Val Thr Asn Val Val Ser Gly Leu Lys Lys Phe 1845 1850 1855 Gly Asp Gly His Thr Leu Ala Asn Gly Thr Val Ala Asp Phe Glu Lys 1860 1865 1870 His Tyr Asp Asn Ala Tyr Lys Asp Leu Thr Asn Leu Asp Glu Lys Gly 1875 1880 1885 Ala Asp Asn Asn Pro Thr Val Ala Asp Asn Thr Ala Ala Thr Val Gly 1890 1895 1900 Asp Leu Arg Gly Leu Gly Trp Val Ile Ser Ala Asp Lys Thr Thr Gly 1905 1910 1915 1920 Glu Pro Asn Gln Glu Tyr Asn Ala Gln Val Arg Asn Ala Asn Glu Val 1925 1930 1935 Lys Phe Lys Ser Gly Asn Gly Ile Asn Val Ser Gly Lys Thr Leu Asn 1940 1945 1950 Gly Thr Arg Val Ile Thr Phe Glu Leu Ala Lys Gly Glu Val Val Lys 1955 1960 1965 Ser Asn Glu Phe Thr Val Lys Asn Ala Asp Gly Ser Glu Thr Asn Leu 1970 1975 1980 Val Lys Val Gly Asp Met Tyr Tyr Ser Lys Glu Asp Ile Asp Pro Ala 1985 1990 1995 2000 Thr Ser Lys Pro Met Thr Gly Lys Thr Glu Lys Tyr Lys Val Glu Asn 2005 2010 2015 Gly Lys Val Val Ser Ala Asn Gly Ser Lys Thr Glu Val Thr Leu Thr 2020 2025 2030 Asn Lys Gly Ser Gly Tyr Val Thr Gly Asn Gln Val Ala Asp Ala Ile 2035 2040 2045 Ala Lys Ser Gly Phe Glu Leu Gly Leu Ala Asp Ala Ala Glu Ala Glu 2050 2055 2060 Lys Ala Phe Ala Glu Ser Ala Lys Asp Lys Gln Leu Ser Lys Asp Lys 2065 2070 2075 2080 Ala Glu Thr Val Asn Ala His Asp Lys Val Arg Phe Ala Asn Gly Leu 2085 2090 2095 Asn Thr Lys Val Ser Ala Ala Thr Val Glu Ser Thr Asp Ala Asn Gly 2100 2105 2110 Asp Lys Val Thr Thr Thr Phe Val Lys Thr Asp Val Glu Leu Pro Leu 2115 2120 2125 Thr Gln Ile Tyr Asn Thr Asp Ala Asn Gly Asn Lys Ile Val Lys Lys 2130 2135 2140 Ala Asp Gly Lys Trp Tyr Glu Leu Asn Ala Asp Gly Thr Ala Ser Asn 2145 2150 2155 2160 Lys Glu Val Thr Leu Gly Asn Val Asp Ala Asn Gly Lys Lys Val Val 2165 2170 2175 Lys Val Thr Glu Asn Gly Ala Asp Lys Trp Tyr Tyr Thr Asn Ala Asp 2180 2185 2190 Gly Ala Ala Asp Lys Thr Lys Gly Glu Val Ser Asn Asp Lys Val Ser 2195 2200 2205 Thr Asp Glu Lys His Val Val Arg Leu Asp Pro Asn Asn Gln Ser Asn 2210 2215 2220 Gly Lys Gly Val Val Ile Asp Asn Val Ala Asn Gly Glu Ile Ser Ala 2225 2230 2235 2240 Thr Ser Thr Asp Ala Ile Asn Gly Ser Gln Leu Tyr Ala Val Ala Lys 2245 2250 2255 Gly Val Thr Asn Leu Ala Gly Gln Val Asn Asn Leu Glu Gly Lys Val 2260 2265 2270 Asn Lys Val Gly Lys Arg Ala Asp Ala Gly Thr Ala Ser Ala Leu Ala 2275 2280 2285 Ala Ser Gln Leu Pro Gln Ala Thr Met Pro Gly Lys Ser Met Val Ala 2290 2295 2300 Ile Ala Gly Ser Ser Tyr Gln Gly Gln Asn Gly Leu Ala Ile Gly Val 2305 2310 2315 2320 Ser Arg Ile Ser Asp Asn Gly Lys Val Ile Ile Arg Leu Ser Gly Thr 2325 2330 2335 Thr Asn Ser Gln Gly Lys Thr Gly Val Ala Ala Gly Val Gly Tyr Gln 2340 2345 2350 Trp 658 amino acids amino acid unknown unknown protein unknown 5 Met Asn Lys Ile Phe Asn Val Ile Trp Asn Val Val Thr Gln Thr Trp 1 5 10 15 Val Val Val Ser Glu Leu Thr Arg Thr His Thr Lys Cys Ala Ser Ala 20 25 30 Thr Val Ala Val Ala Val Leu Ala Thr Leu Leu Ser Ala Thr Val Glu 35 40 45 Ala Asn Asn Asn Thr Pro Val Thr Asn Lys Leu Lys Ala Tyr Gly Asp 50 55 60 Ala Asn Phe Asn Phe Thr Asn Asn Ser Ile Ala Asp Ala Glu Lys Gln 65 70 75 80 Val Gln Glu Ala Tyr Lys Gly Leu Leu Asn Leu Asn Glu Lys Asn Ala 85 90 95 Ser Asp Lys Leu Leu Val Glu Asp Asn Thr Ala Ala Thr Val Gly Asn 100 105 110 Leu Arg Lys Leu Gly Trp Val Leu Ser Ser Lys Asn Gly Thr Arg Asn 115 120 125 Glu Lys Ser Gln Gln Val Lys His Ala Asp Glu Val Leu Phe Glu Gly 130 135 140 Lys Gly Gly Val Gln Val Thr Ser Thr Ser Glu Asn Gly Lys His Thr 145 150 155 160 Ile Thr Phe Ala Leu Ala Lys Asp Leu Gly Val Lys Thr Ala Thr Val 165 170 175 Ser Asp Thr Leu Thr Ile Gly Gly Gly Ala Ala Ala Gly Ala Thr Thr 180 185 190 Thr Pro Lys Val Asn Val Thr Ser Thr Thr Asp Gly Leu Lys Phe Ala 195 200 205 Lys Asp Ala Ala Gly Ala Asn Gly Asp Thr Thr Val His Leu Asn Gly 210 215 220 Ile Gly Ser Thr Leu Thr Asp Thr Leu Val Gly Ser Pro Ala Thr His 225 230 235 240 Ile Asp Gly Gly Asp Gln Ser Thr His Tyr Thr Arg Ala Ala Ser Ile 245 250 255 Lys Asp Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Ala Gly 260 265 270 Ser Thr Thr Gly Gln Ser Glu Asn Val Asp Phe Val His Thr Tyr Asp 275 280 285 Thr Val Glu Phe Leu Ser Ala Asp Thr Glu Thr Thr Thr Val Thr Val 290 295 300 Asp Ser Lys Glu Asn Gly Lys Arg Thr Glu Val Lys Ile Gly Ala Lys 305 310 315 320 Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu Phe Thr Gly Lys Ala 325 330 335 Asn Lys Glu Thr Asn Lys Val Asp Gly Ala Asn Ala Thr Glu Asp Ala 340 345 350 Asp Glu Gly Lys Gly Leu Val Thr Ala Lys Asp Val Ile Asp Ala Val 355 360 365 Asn Lys Thr Gly Trp Arg Ile Lys Thr Thr Asp Ala Asn Gly Gln Asn 370 375 380 Gly Asp Phe Ala Thr Val Ala Ser Gly Thr Asn Val Thr Phe Ala Ser 385 390 395 400 Gly Asn Gly Thr Thr Ala Thr Val Thr Asn Gly Thr Asp Gly Ile Thr 405 410 415 Val Lys Tyr Asp Ala Lys Val Gly Asp Gly Leu Lys Leu Asp Gly Asp 420 425 430 Lys Ile Ala Ala Asp Thr Thr Ala Leu Thr Val Asn Asp Gly Lys Asn 435 440 445 Ala Asn Asn Pro Lys Gly Lys Val Ala Asp Val Ala Ser Thr Asp Glu 450 455 460 Lys Lys Leu Val Thr Ala Lys Gly Leu Val Thr Ala Leu Asn Ser Leu 465 470 475 480 Ser Trp Thr Thr Thr Ala Ala Glu Ala Asp Gly Gly Thr Leu Asp Gly 485 490 495 Asn Ala Ser Glu Gln Glu Val Lys Ala Gly Asp Lys Val Thr Phe Lys 500 505 510 Ala Gly Lys Asn Leu Lys Val Lys Gln Glu Gly Ala Asn Phe Thr Tyr 515 520 525 Ser Leu Gln Asp Ala Leu Thr Gly Leu Thr Ser Ile Thr Leu Gly Thr 530 535 540 Gly Asn Asn Gly Ala Lys Thr Glu Ile Asn Lys Asp Gly Leu Thr Ile 545 550 555 560 Thr Pro Ala Asn Gly Ala Gly Ala Asn Asn Ala Asn Thr Ile Ser Val 565 570 575 Thr Lys Asp Gly Ile Ser Ala Gly Gly Gln Ser Val Lys Asn Val Val 580 585 590 Ser Gly Leu Lys Lys Phe Gly Asp Ala Asn Phe Asp Pro Leu Thr Ser 595 600 605 Ser Ala Asp Asn Leu Thr Lys Gln Asn Asp Asp Ala Tyr Lys Gly Leu 610 615 620 Thr Asn Leu Asp Glu Lys Gly Thr Asp Lys Gln Thr Pro Val Val Ala 625 630 635 640 Asp Asn Thr Ala Ala Thr Val Gly Asp Leu Arg Gly Leu Gly Trp Val 645 650 655 Ile Ser 607 amino acids amino acid unknown unknown protein unknown 6 Met Asn Lys Ile Phe Asn Val Ile Trp Asn Val Met Thr Gln Thr Trp 1 5 10 15 Val Val Val Ser Glu Leu Thr Arg Thr His Thr Lys Arg Leu Arg Asn 20 25 30 Arg Gly Asp Pro Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln Ala 35 40 45 Asn Ala Thr Asp Glu Asp Glu Glu Leu Asp Pro Val Val Arg Thr Ala 50 55 60 Pro Val Leu Ser Phe His Ser Asp Lys Glu Gly Thr Gly Glu Lys Glu 65 70 75 80 Val Thr Glu Asn Ser Asn Trp Gly Ile Tyr Phe Asp Asn Lys Gly Val 85 90 95 Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu Lys Xaa 100 105 110 Lys Gln Xaa Thr Asp Glu Xaa Thr Asn Ala Ser Ser Phe Thr Tyr Ser 115 120 125 Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Ala Thr Glu Lys Leu 130 135 140 Ser Phe Gly Ala Asn Gly Asp Lys Val Asp Ile Thr Ser Asp Ala Asn 145 150 155 160 Gly Leu Lys Leu Ala Lys Thr Gly Asn Gly Asn Val His Leu Asn Gly 165 170 175 Leu Asp Ser Thr Leu Pro Asp Ala Val Thr Asn Thr Gly Val Leu Ser 180 185 190 Ser Ser Ser Phe Thr Pro Asn Asp Val Glu Lys Thr Arg Ala Ala Thr 195 200 205 Val Lys Asp Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Ala Lys Thr 210 215 220 Ala Gly Gly Asn Val Glu Ser Val Asp Leu Val Ser Ala Tyr Asn Asn 225 230 235 240 Val Glu Phe Ile Thr Gly Asp Lys Asn Thr Leu Asp Val Val Leu Thr 245 250 255 Ala Lys Glu Asn Xaa Lys Thr Thr Glu Val Lys Phe Thr Pro Lys Thr 260 265 270 Ser Val Ile Lys Glu Lys Asp Gly Lys Leu Phe Thr Gly Lys Glu Asn 275 280 285 Asn Asp Thr Asn Lys Val Thr Ser Asn Thr Ala Thr Asp Asn Thr Asp 290 295 300 Glu Gly Asn Gly Leu Val Thr Ala Lys Ala Val Ile Asp Ala Val Asn 305 310 315 320 Lys Ala Gly Trp Arg Val Lys Thr Thr Thr Ala Asn Gly Gln Asn Gly 325 330 335 Asp Phe Ala Thr Val Ala Ser Gly Thr Asn Val Thr Phe Glu Ser Gly 340 345 350 Asp Gly Thr Thr Ala Ser Val Thr Lys Asp Thr Asn Gly Asn Gly Ile 355 360 365 Thr Val Lys Tyr Asp Ala Lys Val Gly Asp Gly Leu Lys Phe Asp Ser 370 375 380 Asp Lys Lys Ile Val Ala Asp Thr Thr Ala Leu Thr Val Thr Gly Gly 385 390 395 400 Lys Val Ala Glu Ile Ala Lys Glu Asp Asp Lys Lys Lys Leu Val Asn 405 410 415 Ala Gly Asp Leu Val Thr Ala Leu Gly Asn Leu Ser Trp Lys Ala Lys 420 425 430 Ala Glu Ala Asp Thr Asp Gly Ala Leu Glu Gly Ile Ser Lys Asp Gln 435 440 445 Glu Val Lys Ala Gly Glu Thr Val Thr Phe Lys Ala Gly Lys Asn Leu 450 455 460 Lys Val Lys Gln Asp Gly Ala Asn Phe Thr Tyr Ser Leu Gln Asp Ala 465 470 475 480 Leu Thr Gly Leu Thr Ser Ile Thr Leu Gly Gly Thr Thr Asn Gly Gly 485 490 495 Asn Asp Ala Lys Thr Val Ile Asn Lys Asp Gly Leu Thr Ile Thr Pro 500 505 510 Ala Gly Asn Gly Gly Thr Thr Gly Thr Asn Thr Ile Ser Val Thr Lys 515 520 525 Asp Gly Ile Lys Ala Gly Asn Lys Ala Ile Thr Asn Val Ala Ser Gly 530 535 540 Leu Arg Ala Tyr Asp Asp Ala Asn Phe Asp Val Leu Asn Asn Ser Ala 545 550 555 560 Thr Asp Leu Asn Arg His Val Glu Asp Ala Tyr Lys Gly Leu Leu Asn 565 570 575 Leu Asn Glu Lys Asn Ala Asn Lys Gln Pro Leu Val Thr Asp Ser Thr 580 585 590 Ala Ala Thr Val Gly Asp Leu Arg Lys Leu Gly Trp Val Val Ser 595 600 605 24 amino acids amino acid unknown unknown protein unknown 7 Met Asn Lys Ile Phe Asn Val Ile Trp Asn Val Met Thr Gln Thr Trp 1 5 10 15 Val Val Val Ser Glu Leu Thr Arg 20 24 amino acids amino acid unknown unknown protein unknown 8 Met Asn Lys Ile Phe Asn Val Ile Trp Asn Val Val Thr Gln Thr Trp 1 5 10 15 Val Val Val Ser Glu Leu Thr Arg 20 24 amino acids amino acid unknown unknown protein unknown 9 Met Asn Lys Ile Tyr Arg Leu Lys Phe Ser Lys Arg Leu Asn Ala Leu 1 5 10 15 Val Ala Val Ser Glu Leu Ala Arg 20 24 amino acids amino acid unknown unknown protein unknown 10 Met Asn Lys Ile Tyr Arg Leu Lys Phe Ser Lys Arg Leu Asn Ala Leu 1 5 10 15 Val Ala Val Ser Glu Leu Ala Arg 20 24 amino acids amino acid unknown unknown protein unknown 11 Met Asn Lys Ala Tyr Ser Ile Ile Trp Ser His Ser Arg Gln Ala Trp 1 5 10 15 Ile Val Ala Ser Glu Leu Ala Arg 20 24 amino acids amino acid unknown unknown protein unknown 12 Met Asn Arg Ile Tyr Ser Leu Arg Tyr Ser Ala Val Ala Arg Gly Phe 1 5 10 15 Ile Ala Val Ser Glu Phe Ala Arg 20 24 amino acids amino acid unknown unknown protein unknown 13 Met Asn Lys Ile Tyr Tyr Leu Lys Tyr Cys His Ile Thr Lys Ser Leu 1 5 10 15 Ile Ala Val Ser Glu Leu Ala Arg 20 2037 base pairs nucleic acid unknown unknown DNA unknown 14 ATGAACAAAA TTTTTAACGT TATTTGGAAT GTTGTGACTC AAACTTGGGT TGTCGTATCT 60 GAACTCACTC GCACCCACAC CAAATGCGCC TCCGCCACCG TGGCAGTTGC CGTATTGGCA 120 ACCCTGTTGT CCGCAACGGT TCAGGCGAAT GCTACCGATG AAAACGAAGA TGATGAAGAA 180 GAGTTAGAAC CCGTACAACG CTCTGTTTTA AGGTGGAGCT TCAAATCCGC TAAGGAAGGC 240 ACTGGAGAAC AAGAGGGAAC AACAGAGGTA ATAAATTTGA ACACAGATTC ATCAGGAAAT 300 GCAGTAGGAA GCAGCACAAT CACCTTCAAA GCCGGCGACA ACCTGAAAAT CAAACAAAGC 360 GGCAATGACT TCACCTACTC GCTGAAAAAA GAGCTGAAAA ACCTGACCAG TGTTGAAACT 420 GAAAAATTAT CGTTTGGCGC AAACGGCAAT AAAGTTGATA TTACCAGTGA TGCAAATGGC 480 TTGAAATTGG CGAAAACAGG TAACGGAAAT GGTCAAAACA GTAATGTTCA CTTAAACGGT 540 ATTGCTTCGA CTTTGACCGA TACGCTTGCC GGTGGCACAA CAGGACACGT TGACACCAAC 600 ATTGATGCGG TTAATTATCA TCGCGCTGCA AGCGTACAAG ATGTGTTAAA CAGCGGTTGG 660 AATATCCAAG GCAATGGAAA CAATGTCGAT TTTGTCCGTA CTTACGACAC CGTGGACTTT 720 GTCAATGGCG CGAATGCCAA TGTGAGCGTT ACGGCTGATA CGGCTCACAA AAAGACAACT 780 GTCCGTGTGG ATGTAACAGG CTTGCCGGTT CAATATGTTA CGGAAGACGG CAAAACCGTT 840 GTGAAAGTGG GCAATGAGTA TTACAAAGCC AAAGATGACG GTTCGGCGGA TATGAATCAA 900 AAAGTCGAAA ACGGCGAGCT GGCGAAAACC AAAGTGAAAT TGGTATCGGC AAGCGGTACA 960 AATCCGGTGA AAATTAGCAA TGTTGCAGAC GGCACGGAAG ACACCGATGC GGTCAGCTTT 1020 AAGCAATTAA AAGCCTTGCA AGACAAACAG GTTACGTTGA GCACGAGCAA TGCTTATGCC 1080 AATGGCGGTA CAGATAACGA CGGCGGCAAG GCAACTCAAA CTTTAAGCAA TGGTTTGAAT 1140 TTTAAATTTA AATCTAGCGA TGGCGAGTTG TTGAAAATTA GCGCGACCGG CGATACGGTT 1200 ACTTTTACGC CGAAAAAAGG TTCGGTACAG GTTGGCGATG ATGGCAAGGC TTCAATTTCA 1260 AAAGGTGCAA ATACAACTGA AGGTTTGGTT GAGGCTTCTG AATTGGTTGA AAGCCTGAAC 1320 AAACTGGGTT GGAAAGTAGG GGTTGAGAAA GTCGGCAGCG GCGAGCTTGA TGGTACATCC 1380 AAGGAAACTT TAGTGAAGTC GGGCGATAAA GTAACTTTGA AAGCCGGCGA CAATCTGAAG 1440 GTCAAACAAG AGGGCACAAA CTTCACTTAC GCGCTCAAAG ATGAATTGAC GGGCGTGAAG 1500 AGCGTGGAGT TTAAAGACAC GGCGAATGGT GCAAACGGTG CAAGCACGAA GATTACCAAA 1560 GACGGCTTGA CCATTACGCT GGCAAACGGT GCGAATGGTG CGACGGTGAC TGATGCCGAC 1620 AAGATTAAAG TTGCTTCGGA CGGCATTAGC GCGGGTAATA AAGCAGTTAA AAACGTCGCG 1680 GCAGGCGAAA TTTCTGCCAC TTCCACCGAT GCGATTAACG GAAGCCAGTT GTATGCCGTG 1740 GCAAAAGGGG TAACAAACCT TGCTGGACAA GTGAATAATC TTGAGGGCAA AGTGAATAAA 1800 GTGGGCAAAC GTGCAGATGC AGGTACTGCA AGTGCATTAG CGGCTTCACA GTTACCACAA 1860 GCCACTATGC CAGGTAAATC AATGGTTTCT ATTGCGGGAA GTAGTTATCA AGGTCAAAAT 1920 GGTTTAGCTA TCGGGGTATC AAGAATTTCC GATAATGGCA AAGTGATTAT TCGCTTGTCT 1980 GGCACAACCA ATAGTCAAGG TAAAACAGGC GTTGCAGCAG GTGTTGGTTA CCAGTGG 2037 679 amino acids amino acid unknown unknown protein unknown 15 Met Asn Lys Ile Phe Asn Val Ile Trp Asn Val Val Thr Gln Thr Trp 1 5 10 15 Val Val Val Ser Glu Leu Thr Arg Thr His Thr Lys Cys Ala Ser Ala 20 25 30 Thr Val Ala Val Ala Val Leu Ala Thr Leu Leu Ser Ala Thr Val Gln 35 40 45 Ala Asn Ala Thr Asp Glu Asn Glu Asp Asp Glu Glu Glu Leu Glu Pro 50 55 60 Val Gln Arg Ser Val Leu Arg Trp Ser Phe Lys Ser Ala Lys Glu Gly 65 70 75 80 Thr Gly Glu Gln Glu Gly Thr Thr Glu Val Ile Asn Leu Asn Thr Asp 85 90 95 Ser Ser Gly Asn Ala Val Gly Ser Ser Thr Ile Thr Phe Lys Ala Gly 100 105 110 Asp Asn Leu Lys Ile Lys Gln Ser Gly Asn Asp Phe Thr Tyr Ser Leu 115 120 125 Lys Lys Glu Leu Lys Asn Leu Thr Ser Val Glu Thr Glu Lys Leu Ser 130 135 140 Phe Gly Ala Asn Gly Asn Lys Val Asp Ile Thr Ser Asp Ala Asn Gly 145 150 155 160 Leu Lys Leu Ala Lys Thr Gly Asn Gly Asn Gly Gln Asn Ser Asn Val 165 170 175 His Leu Asn Gly Ile Ala Ser Thr Leu Thr Asp Thr Leu Ala Gly Gly 180 185 190 Thr Thr Gly His Val Asp Thr Asn Ile Asp Ala Val Asn Tyr His Arg 195 200 205 Ala Ala Ser Val Gln Asp Val Leu Asn Ser Gly Trp Asn Ile Gln Gly 210 215 220 Asn Gly Asn Asn Val Asp Phe Val Arg Thr Tyr Asp Thr Val Asp Phe 225 230 235 240 Val Asn Gly Ala Asn Ala Asn Val Ser Val Thr Ala Asp Thr Ala His 245 250 255 Lys Lys Thr Thr Val Arg Val Asp Val Thr Gly Leu Pro Val Gln Tyr 260 265 270 Val Thr Glu Asp Gly Lys Thr Val Val Lys Val Gly Asn Glu Tyr Tyr 275 280 285 Lys Ala Lys Asp Asp Gly Ser Ala Asp Met Asn Gln Lys Val Glu Asn 290 295 300 Gly Glu Leu Ala Lys Thr Lys Val Lys Leu Val Ser Ala Ser Gly Thr 305 310 315 320 Asn Pro Val Lys Ile Ser Asn Val Ala Asp Gly Thr Glu Asp Thr Asp 325 330 335 Ala Val Ser Phe Lys Gln Leu Lys Ala Leu Gln Asp Lys Gln Val Thr 340 345 350 Leu Ser Thr Ser Asn Ala Tyr Ala Asn Gly Gly Thr Asp Asn Asp Gly 355 360 365 Gly Lys Ala Thr Gln Thr Leu Ser Asn Gly Leu Asn Phe Lys Phe Lys 370 375 380 Ser Ser Asp Gly Glu Leu Leu Lys Ile Ser Ala Thr Gly Asp Thr Val 385 390 395 400 Thr Phe Thr Pro Lys Lys Gly Ser Val Gln Val Gly Asp Asp Gly Lys 405 410 415 Ala Ser Ile Ser Lys Gly Ala Asn Thr Thr Glu Gly Leu Val Glu Ala 420 425 430 Ser Glu Leu Val Glu Ser Leu Asn Lys Leu Gly Trp Lys Val Gly Val 435 440 445 Glu Lys Val Gly Ser Gly Glu Leu Asp Gly Thr Ser Lys Glu Thr Leu 450 455 460 Val Lys Ser Gly Asp Lys Val Thr Leu Lys Ala Gly Asp Asn Leu Lys 465 470 475 480 Val Lys Gln Glu Gly Thr Asn Phe Thr Tyr Ala Leu Lys Asp Glu Leu 485 490 495 Thr Gly Val Lys Ser Val Glu Phe Lys Asp Thr Ala Asn Gly Ala Asn 500 505 510 Gly Ala Ser Thr Lys Ile Thr Lys Asp Gly Leu Thr Ile Thr Leu Ala 515 520 525 Asn Gly Ala Asn Gly Ala Thr Val Thr Asp Ala Asp Lys Ile Lys Val 530 535 540 Ala Ser Asp Gly Ile Ser Ala Gly Asn Lys Ala Val Lys Asn Val Ala 545 550 555 560 Ala Gly Glu Ile Ser Ala Thr Ser Thr Asp Ala Ile Asn Gly Ser Gln 565 570 575 Leu Tyr Ala Val Ala Lys Gly Val Thr Asn Leu Ala Gly Gln Val Asn 580 585 590 Asn Leu Glu Gly Lys Val Asn Lys Val Gly Lys Arg Ala Asp Ala Gly 595 600 605 Thr Ala Ser Ala Leu Ala Ala Ser Gln Leu Pro Gln Ala Thr Met Pro 610 615 620 Gly Lys Ser Met Val Ser Ile Ala Gly Ser Ser Tyr Gln Gly Gln Asn 625 630 635 640 Gly Leu Ala Ile Gly Val Ser Arg Ile Ser Asp Asn Gly Lys Val Ile 645 650 655 Ile Arg Leu Ser Gly Thr Thr Asn Ser Gln Gly Lys Thr Gly Val Ala 660 665 670 Ala Gly Val Gly Tyr Gln Trp 675 21 base pairs nucleic acid unknown unknown DNA unknown 16 CCGTGCTTGC CCAACACGCT T 21 21 base pairs nucleic acid unknown unknown DNA unknown 17 GCTGCCACCT TGCACAACAA C 21 21 base pairs nucleic acid unknown unknown DNA unknown 18 CTTTCAATGC CAGAAAGTAG G 21 21 base pairs nucleic acid unknown unknown DNA unknown 19 CTTCAACCGT TGCGGACAAC A 21 

What is claimed is:
 1. An isolated polypeptide comprising an immunogenic epitope of a recombinant Haemophilus adhesion protein selected from the group conk of the sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:4) and FIG. 15 (SEQ ID NO:15).
 2. The isolated polypeptide according to claim 1 having greater than 90% identity to a sequence selected from the group consisting of the sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:4) and FIG. 15 (SEQ ID NO:15).
 3. The isolated polypeptide according to claim 2 which has the sequence shown in FIG. 3 (SEQ ID NO:4).
 4. The isolated polypeptide according to claim 2 which has the sequence shown in FIG. 15 (SEQ ID NO:15).
 5. The isolated polypeptide according to claim 2 which has the sequence shown in FIG. 2 (SEQ ID NO:2).
 6. An immunogenic composition comprising a pharmaceutically acceptable carrier and the isolated polypeptide of claim 1 for use in generating an immune response.
 7. An immunogenic composition according to claim 6 wherein said isolated polypeptide is encoded by a recombinant nucleic acid encoding a Haemphilus adhesion protein.
 8. A method of manufacturing a medicament comprising the composition of claim 6, said method comprising: (a) culturing a host cell transformed with nucleic acid encoding a Haemophilus adhesion protein: (b) expressing said nucleic acid to produce said Haemophilus adhesion protein; and (c) admixing said Haemophilus adhesion protein with a pharmaceutically acceptable carrier.
 9. A method of manufacturing a medicament according to claim 8 wherein said Haemophilus adhesion protein is isolated from a non-typable H. influenzae.
 10. The isolated polypeptide according to claim 2 having greater than 90% identity to the sequence shown in FIG. 2 (SEQ ID NO:2).
 11. The isolated polypeptide according to claim 2 having greater than 90% identity to the sequence shown in FIG. 3 (SEQ ID NO.4).
 12. The isolated polypeptide according to claim 2 having greater than 90% identity to the sequence shown in FIG. 15 (SEQ ID NO:15).
 13. The isolated polypeptide according to claim 2 encoded by a nucleic acid which will hybridize under high stringency conditions to the nucleic acid shown in FIG. 14 (SEQ ID NO:14).
 14. The isolated polypeptide according to claim 2 encoded by a nucleic acid which will hybridize under high stringency conditions to the nucleic acid shown in FIG. 1 (SEQ ID NO:1).
 15. The isolated polypeptide according to claim 2 encoded by a nucleic acid which will hybridize under high stringency conditions to the nucleic acid shown in FIG. 3 (SEQ ID NO:3).
 16. A recombinant Haemophilus adhesion protein encoded by a nucleic acid sequence selected from the group consisting of the nucleic acid sequence as shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:3) and FIG. 14 (SEQ ID NO:14).
 17. The recombinant Haemophilus adhesion of claim 16 wherein said nucleic acid sequence comprises the nucleic acid sequence as shown in FIG. 1 (SEQ ID NO:1).
 18. The recombinant Haemophilus adhesion protein of claim 16 wherein said nucleic acid sequence comprises the nucleic acid sequence as shown in FIG. 3 (SEQ ID NO:3).
 19. The recombinant Haemophilus adhesion protein of claim 16 wherein said nucleic acid sequence comprises the nucleic acid sequence as shown in FIG. 14 (SEQ ID NO:14).
 20. A composition comprising a pharmaceutically acceptable carrier and the isolated polypeptide according to any one of claims 2, 10, 11 or
 12. 